Novel Class of Monospecific and Bispecific Humanized Antibodies that Target the Insulin-like Growth Factor Type I Receptor (IGF-1R)

ABSTRACT

The present invention provides compositions and methods of use of anti-IGF-1R antibodies or antibody fragments. Preferably the antibodies bind to IGF-1R but not IR; are not agonists for IGF-1R; do not block binding of IGF-1 or IGF-2 to isolated IGF-1R, but effectively neutralize activation of IGF-1R by IGF-1 in intact cells; and block binding of an R1 antibody to IGF-1R. The antibodies may be murine, chimeric, humanized or human R1 antibodies comprising the heavy chain CDR sequences DYYMY (SEQ ID NO:1), YITNYGGSTYYPDTVKG (SEQ ID NO:2) and QSNYDYDGWFAY (SEQ ID NO:3) and the light chain CDR sequences KASQEVGTAVA (SEQ ID NO:4), WASTRHT (SEQ ID NO:5) and QQYSNYPLT (SEQ ID NO:6). Preferably the antibodies bind to an epitope of IGF-1R comprising the first half of the cysteine-rich domain of IGF-1R (residues 151-222). The anti-IGF-1R antibodies may be used for diagnosis or therapy of various diseases such as cancer.

FIELD OF THE INVENTION

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 12/689,336, filed Jan. 19, 2010, which claimed thebenefit under 35 U.S.C. 119(e) of Provisional U.S. Patent ApplicationSer. No. 61/145,896, filed Jan. 20, 2009, each priority applicationincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 9, 2010, is namedIMM31US2.txt, and is 19,199 bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies and antigen-binding antibodyfragments that bind to the insulin-like growth factor type I receptor(IGF-1R), but not to the insulin receptor (IR). In preferredembodiments, the anti-IGF-1R antibody is not an agonist for IGF-1R. Inmore preferred embodiments, the anti-IGF-1R antibody binds to an epitopeof IGF-1R comprising the first half of the cysteine-rich domain ofIGF-1R, between amino acid residues 151 and 222. In most preferredembodiments, the anti-IGF-1R antibody does not block binding of IGF-1 orIGF-2 to isolated IGF-1R, but effectively neutralizes the activation ofIGF-1R by IGF-1 in situ in intact cells or tissues. In otherembodiments, the mouse anti-IGF-1R antibody, designated as R1, comprisesthe heavy chain variable region complementarity determining region (CDR)sequences CDR1 (DYYMY, SEQ ID NO:1), CDR2 (YITNYGGSTYYPDTVKG, SEQ IDNO:2) and CDR3 (QSNYDYDGWFAY, SEQ ID NO:3) and the light chain variableregion CDR sequences CDR1 (KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQID NO:5) and CDR3 (QQYSNYPLT, SEQ ID NO:6). In more preferredembodiments, the anti-IGF-1R antibody is a humanized, chimeric or humanR1 antibody, designated as hR1, comprising the CDR sequences recitedabove. In most preferred embodiments, the anti-IGF-R1 antibody is ahumanized antibody comprising the recited CDR sequences and humanantibody constant and framework (FR) region sequences.

Such antibodies and fragments are of use for detection and/or therapy ofa wide variety of cancers where IGF-1R expression is important forcancer cell transformation, growth, survival, metastasis or resistanceto other therapeutic agents, including but not limited to Wilms' tumor,Ewing sarcoma, neuroblastoma, neuroendocrine tumors, melanoma,glioblastomas, skin, breast, head-and-neck, colon, rectal, gastric,esophageal, ovarian, bladder, prostate, liver, renal, pancreatic and/orlung cancers, as well as lymphomas, leukemias, and myelomas. Theanti-IGF-1R antibodies and/or antibody fragments may be used incompositions and therapeutic methods either alone or in conjunction withother cytotoxic agents such as cancer chemotherapeutic agents,pro-apoptotic agents, radionuclides, EGFR inhibitors (e.g. erlotinib oranti-EGFR antibodies), anti-angiogenesis agents (e.g., anti-VEGF andanti-PIGF peptides or antibodies) and/or other IGF-1R inhibitors such astryphostins (e.g., AG1024, AG538), pyrrolo[2,3-d]-pyrimidine derivatives(e.g., NVP-AEW541) or other anti-IGF-1R antibodies or antibodies againstother tumor-associated antigens (TAA). The anti-IGF-1R antibodies may benaked antibodies or may be conjugated to one or more therapeutic and/ordiagnostic agents. The antibodies may be murine, chimeric, humanized orhuman anti-IGF-1R antibodies.

Other embodiments may relate to multispecific antibodies, bispecificantibodies, antibody fusion proteins or fragments thereof comprising atleast one anti-IGF-1R monoclonal antibody (MAb) or fragment thereof, insome cases in combination with a second, different antibody or fragment.The antibodies, fragments or antibody fusion proteins may beadministered alone, as a therapeutic immunoconjugate or in combinationwith one or more therapeutic agents, with other naked antibodies orother immunoconjugates. Still other embodiments relate to DNA sequencesencoding anti-IGF-1R antibodies or antibody fusion proteins, vectors andhost cells containing the DNA sequences, and methods of making theanti-IGF-1R antibodies. Further embodiments concern multivalent,multispecific and/or multifunctional constructs made by thedock-and-lock (DNL) technique that incorporate anti-IGF-1R antibodies,fusion proteins and/or fragments thereof.

2. Related Art

The insulin-like growth factor type I receptor (IGF-1R) is a member ofthe large class of tyrosine kinase receptors, which regulate a varietyof intracellular pathways. IGF-1R binds IGF-1, a polypeptide hormonestructurally similar to insulin (Laron, Mol. Pathol. 2001, 54:311-16).The IGF-1 receptor is homologous to the insulin receptor (IR), sharingabout 70% overall sequence homology with IR (Riedemann and Macaulay,Endocrine-Related Cancer, 2006, 13:S33-43). Not surprisingly, inhibitorsdeveloped against IGF-1R tend to show cross-reactivity with the insulinreceptor, accounting for at least part of the toxicity profiles of suchcompounds (Miller and Yee, 2005, Cancer Res. 65:10123-27; Riedemann andMacaulay, 2006).

The IGF system plays an important role in regulating cell proliferation,differentiation, apoptosis and transformation (Jones et al,Endocrinology Rev. 1995. 16:3-34). The IGF system comprises tworeceptors, insulin like growth factor receptor 1 (IGF-1R; CD221) andinsulin like growth factor receptor 2 (IGF-2R; CD222); two ligands,insulin like growth factor 1 (IGF-1) and IGF-2; and several IGF bindingproteins (IGFBP-1 to IGFBP-6). In addition, a large group of IGFBPproteases (e.g., caspases, metalloproteinases, prostate-specificantigen) hydrolyze IGF bound IGFBP to release free IGFs, which theninteract with IGF-1R and IGF-2R.

IGF-1R comprises two extracellular α subunits (130-135 kD) and twomembrane spanning β-subunits (95 kD) that contain the cytoplasmictyrosine kinase domain. IGF-1R, like the insulin receptor (IR), differsfrom other receptor tyrosine kinase family members by having a covalentdimeric (α2β2) structure. IGF-1R contains 84% sequence identity to IR inthe kinase domain, while the membrane and C-terminal regions share 61%and 44% sequence identity, respectively (Ulrich et al., EMBO J., 1986,5:2503-12; Blakesley et al., Cytokine Growth Factor Rev., 1996.7:153-56).

IGF-1 and IGF-2 are activating ligands of IGF-1R. Binding of IGF-1 andIGF-2 to the α-chain induces conformational changes that result inautophosphorylation of each β-chain at specific tyrosine residues,converting the receptor from the unphosphorylated inactive state to thephosphorylated active state. The activation of three tyrosine residuesin the activation loop (Tyr residues at 1131, 1135 and 1136) of thekinase domain leads to an increase in catalytic activity that triggersdocking and phosphorylation of substrates such as IRS-1 and Shc adaptorproteins. Activation of these substrates leads to phosphorylation ofadditional proteins involved in the signaling cascade of survival (PI3K,AKT, TOR, S6) and/or proliferation (mitogen-activated protein kinase,p42/p44) (Pollak et al., Nature Reviews Cancer. 2004. 4:505-516; Basergaet al., Biochim Biophys Acta. 1997. 1332:F105-F126; Baserga et al, Int.J. Cancer. 2003. 107:873-77).

IGF-1R has anti-apoptotic effects in both normal and cancer cells(Resnicoff et al., 1995, Cancer Res. 55:2463-69; Kang et al., Am JPhysiol Renal Physiol., 2003, 285:F1013-24; Riedemann and Macaulay,2006). IGF-1R activation has been reported to be significant in thedevelopment of resistance to a variety of cytotoxic agents, such aschemotherapeutic agents, radionuclides and EGFR inhibitors (Jones etal., Endocr Relat Cancer 2004, 11:793-814; Warshamana-Greene et al.,2005, Clin. Cancer Res. 11:1563-71; Riedemann and Macaulay, 2006; Lloretet al., 2007, Gynecol. Oncol. 106:8-11). IGF-1R is overexpressed in awide range of tumor lines, such as melanoma, neuroblastoma, coloncancer, prostate cancer, renal cancer, breast cancer and pancreaticcancer (Ellis et al., 1998, Breast Cancer Treat. 52:175-84; van Golen etal., 2000, Cell Death Differ. 7:654-65; Zhang et al., 2001, BreastCancer Res. 2:170-75; Jones et al., 2004; Riedemann and Macaulay, 2006).A functional IGF-1R is required for transformation and promotes cancercell growth, survival and metastasis (Riedemann and Macaulay, 2006).

Attempts have been made to develop IGF-1R inhibitors for use asanti-cancer agents, such as tyrphostins, pyrrolo[2,3-d]-pyrimidinederivatives, nordihydroguaiaretic acid analogs, diaryureas, AG538,AG1024, NVP-AEW541, NVP-ADW742, BMS-5326924, BMS-554417, OSI-906,INSM-18, luteolin, simvastatin, silibinin, black tea polyphenols,picropodophyllin, anti-IGF-1R antibodies and siRNA inhibitors (Arteagaet al., 1998, J Clin Invest. 84:1418-23; Warshamana-Greene et al., 2005;Klein and Fischer, 2002, Carcinogenesis 23:217-21; Blum et al., 2000,Biochemistry 39:15705-12; Garcia-Echeverria et al., 2004, Cancer Cell5:231-39; Garber, 2005, JNCI97:790-92; Bell et al., 2005, Biochemistry44:930-40; Wu et al., 2005, Clin Cancer Res 11:3065-74; Wang et al.,2005, Mol Cancer Ther 4:1214-21; Singh and Agarwal, 2006, Mol. Carinog.45:436-42; Gable et al., 2006, Mol Cancer Ther 5:1079-86; Niu et al.,Cell Biol Int., 2007, 31:156-64; Blecha et al., 2007, Biorg Med Chem.Lett. 17:4026-29; Qian et al., 2007, Acta Biochim Biophys Sin,39:137-47; Fang et al., 2007, Carcinogenesis 28:713-23; Cohen et al.,2005, Clin Cancer Res 11:2063-73; Sekine et al., Biochem Biophys ResCommun., 2008, 25:356-61; Haluska et al., 2008, J Clin Oncol. 26:May 20suppl; abstr 14510; U.S. Patent Application Publ. No. 2006-233810, theExamples section of each of which is incorporated herein by reference).Typically, these agents have tended to cross-react to a greater orlesser extent with both IGF-1R and IR and/or to act as IGF-1R agonists.The use of such agents for cancer therapy has been limited by theirtoxicity (Riedemann and Macaulay, 2006). A need exists in the field foranti-IGF-1R antibodies that (i) do not cross-react with the insulinreceptor, (ii) exhibit a lower toxicity profile, (iii) neutralize theeffect of IGF-1 and IGF-2 on IGF-1R-expressing cells; (iv) preferably donot act as IGF-1R agonists; and (v) may not compete for binding toisolated IGF-1R with IGF-1 or IGF-2.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods of use ofanti-IGF-1R antibodies or antigen-binding fragments thereof. Inpreferred embodiments, the anti-IGF-1R antibodies bind to IGF-1R but notto IR. In more preferred embodiments the anti-IGF-1R antibodies are notagonists of IGF-1R. In most preferred embodiments, the anti-IGF-1Rantibodies bind to an epitope of IGF-1R comprising the first half of thecysteine-rich domain of IGF-1R, between amino acid residues 151 and 222of the human IGF-1R sequence. (See, e.g., Adams et al., Cell Mol LifeSci 57:1050-93, 2000; NCBI Accession No. AAB22215). Protease cleavage byfurin results in production of the α- chain, comprising residues 1-706,and the 13-chain, comprising residues 711-1337. Residues 151-222consists of the N-terminal half of the cysteine-rich domain (residues151-300).

Preferably, the anti-IGF-1R antibody is a murine, chimeric, humanized orhuman antibody or antigen-binding fragment thereof comprising the heavychain CDR sequences CDR1 (DYYMY, SEQ ID NO:1), CDR2 (YITNYGGSTYYPDTVKG,SEQ ID NO:2) and CDR3 (QSNYDYDGWFAY, SEQ ID NO:3) and the light chainCDR sequences CDR1 (KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQ IDNO:5) and CDR3 (QQYSNYPLT, SEQ ID NO:6). In alternative embodiments, theanti-IGF-1R antibody is a chimeric, humanized or human antibody thatbinds to the same epitope and/or that blocks binding to IGF-1R of amurine R1 antibody comprising the heavy chain CDR sequences CDR1 (DYYMY,SEQ ID NO:1), CDR2 (YITNYGGSTYYPDTVKG, SEQ ID NO:2) and CDR3(QSNYDYDGWFAY, SEQ ID NO:3) and the light chain CDR sequences CDR1(KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQ ID NO:5) and CDR3(QQYSNYPLT, SEQ ID NO:6). The anti-IGF-1R antibody may be a nakedantibody or may be an immunoconjugate attached to at least onetherapeutic agent and/or at least one diagnostic agent.

Various embodiments may concern multispecific antibodies, bispecificantibodies or antibody fusion proteins comprising at least oneanti-IGF-1R MAb or fragment thereof or a first anti-IGF-1R MAb orfragment thereof and a second MAb. Other embodiments may concernpharmaceutical compositions for or methods of use of a first anti-IGF-1RMAb or fragment thereof and a second MAb for therapy of cancer. Thesecond MAb may bind to a tumor-associated antigen (TAA), or a hapten,for example on a targetable construct. A variety of tumor-associatedantigens are known in the art and any such known TAA may targeted by asecond MAb, including but not limited to carbonic anhydrase IX, CCCL19,CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, ED-B offibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1,hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1(ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5,PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR,tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2),VEGFR, EGFR, PIGF, complement factors C3, C3a, C3b, C5a, C5, and anoncogene product.

The second MAb may be selected from any of a wide variety of anti-cancerantibodies known in the art, including but not limited to hPAM4 (U.S.Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No.7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No.7,312,318), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No.7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. ProvisionalPatent Application 61/145,896), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections. The second MAb may also be selected from anyanti-hapten antibody known in the art, including but not limited to h679(U.S. Pat. No. 7,429,381) and 734 (U.S. Pat. No. 7,405,320) or h734, thetext of each of which is incorporated herein by reference. In certainembodiments, a second, different anti-IGF-1R antibody may be used, suchas any of the anti-IGF-1R antibodies in clinical development (see, e.g.,Ryan and Goss, The Oncologist, 2008, 13:16-24).

Other embodiments may concern therapeutic or diagnostic conjugates ofanti-IGF-1R MAbs or fragments thereof or antibody fusion proteins, boundto at least one therapeutic agent or at least one diagnostic agent.Antibodies and fusion proteins with multiple therapeutic agents of thesame or different type are also encompassed. In alternative embodiments,the antibodies, fragments or fusion proteins may be used in therapeuticor diagnostic pre-targeting methods, for example using bispecificantibodies with one arm that binds specifically to a tumor-associatedantigen and a second arm that binds to a targetable construct attachedto one or more diagnostic or therapeutic agents. Methods ofpre-targeting with bispecific antibodies are well known in the art (see,e.g., U.S. Pat. Nos. 7,300,644; 7,138,103; 7,074,405; 7,052,872;6,962,702; 6,458,933, the Examples section of each of which isincorporated herein by reference).

Various embodiments concern methods of using the anti-IGF-1R MAbs orfragments thereof or antibody fusion proteins for therapy or diagnosis,either alone or in combination with one or more other therapeuticagents. The anti-IGF-1R MAb may be used as a naked antibody or as animmunoconjugate attached to one or more therapeutic agents and/ordiagnostic agents. Either naked anti-IGF-1R MAbs or immunoconjugates maybe used in combination therapies administered before, simultaneouslywith or after one or more other therapeutic agents. Any therapeuticagent known in the art, as discussed in more detail below, may beutilized in combination with or attached to an anti-IGF-1R MAb,including but not limited to radionuclides, immunomodulators,anti-angiogenic agents, cytokines, chemokines, growth factors, hormones,drugs, prodrugs, enzymes, oligonucleotides, siRNAs, pro-apoptoticagents, photoactive therapeutic agents, cytotoxic agents,chemotherapeutic agents, toxins, other antibodies or antigen bindingfragments thereof. In preferred embodiments the other therapeutic agentmay be an EGFR inhibitor (e.g., erlotinib or anti-EGFR antibody, such aserbitux) and/or other IGF-1R inhibitors such as tryphostins (e.g.,AG1024, AG538), pyrrolo[2,3-d]-pyrimidine derivatives (e.g., NVP-AEW541)or other anti-IGF-1R antibodies.

Any cancer or diseased cell that expresses IGF-1R may be treated and/ordiagnosed with the anti-IGF-1R antibodies, including but not limited toWilms' tumor, Ewing sarcoma, neuroendocrine tumors, glioblastomas,neuroblastoma, melanoma, skin, breast, colon, rectum, prostate, liver,renal, pancreatic and/or lung cancer, as well as lymphomas, leukemias,and myelomas. Other forms of cancer that may be treated include but arenot limited to acute lymphoblastic leukemia, acute myelogenous leukemia,biliary cancer, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, endometrial cancer, esophageal cancer, gastriccancer, head and neck cancer, Hodgkin's lymphoma, medullary thyroidcarcinoma, non-Hodgkin's lymphoma, ovarian cancer, glioma and urinarybladder cancer.

Certain embodiments may comprise the therapeutic and/or diagnostic useof chimeric, humanized or human R1 antibodies comprising the heavy chainCDR sequences CDR1 (DYYMY, SEQ ID NO:1), CDR2 (YITNYGGSTYYPDTVKG, SEQ IDNO:2) and CDR3 (QSNYDYDGWFAY, SEQ ID NO:3) and the light chain CDRsequences CDR1 (KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQ ID NO:5)and CDR3 (QQYSNYPLT, SEQ ID NO:6). The use of chimeric antibodies ispreferred because they do not elicit as strong a human anti-mouseantibody (HAMA) response as murine antibodies. The use of humanizedantibodies is even more preferred, in order to further reduce thepossibility of inducing a HAMA reaction. As discussed below, techniquesfor humanization of murine antibodies by replacing murine framework andconstant region sequences with corresponding human antibody frameworkand constant region sequences are well known in the art and have beenapplied to numerous murine anti-cancer antibodies. Antibody humanizationmay also involve the substitution of one or more human framework aminoacid residues with the corresponding residues from the parent murineframework region sequences.

Still other embodiments relate to DNA sequences encoding anti-IGF-1Rantibodies or antibody fusion proteins, vectors and host cellscontaining the DNA sequences, and methods of making the anti-IGF-1Rantibodies. In preferred embodiments, the DNA sequences may comprisesequences coding for the hR1VH (SEQ ID NO:9) and hR1VK (SEQ ID NO:10)variable region amino acid sequences. Further embodiments concernmultivalent, multispecific and/or multifunctional constructs made by thedock-and-lock (DNL) technique that incorporate anti-IGF-1R antibodies,fusion proteins and/or fragments thereof. Compositions and methods forproduction and use of DNL constructs have been reported (see, e.g., U.S.Pat. Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787 and U.S. PatentApplication Ser. Nos. 11/925,408, filed Oct. 26, 2007, and 12/418,877,filed Apr. 6, 2009; the Examples section of each of which isincorporated herein by reference).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic diagram of plasmid cRlpdHL2.

FIG. 2. Binding specificity of chimeric R1 (cR1) antibodies toimmobilized recombinant human IGF-1R and recombinant human IR. The cR1was obtained from two different clones -709.2D2 and 710.2G2. The cR1antibodies bind to human IGF-1R but not to the human insulin receptor(IR).

FIG. 3. Binding affinity of cR1 to immobilized recombinant human IGF-1R.

FIG. 4. Competitive binding of murine R1 (ML04R1) and chimeric R1 (cR1)antibodies to immobilized recombinant human IGF-1R.

FIG. 5. Comparison of binding of humanized R1 (hR1), chimeric R1 (cR1)and murine R1 (ML04R1) antibodies to immobilized recombinant humanIF-1R.

FIG. 6. Chimeric R1 (cR1) does not block binding of IGF-1 or IGF-2 toimmobilized recombinant human IGF-1R. ¹²⁵I-labeled IGF-1 or IGF-2 wasincubated with unlabeled IGF-1, IGF-2 or cR1.

FIG. 7. Humanized R1 (hR1) and murine R1 do not block binding of IGF-1to immobilized recombinant human IGF-1R. ¹²⁵I-labeled IGF-1 wasincubated with unlabeled IGF-1, MAb391, hR1 or R1.

FIG. 8. Binding of R1 antibody is not competitive with MAB391. Bindingof fluorescently labeled R1 antibody (A) or MAB391 (B) to immobilizedrhIGF-1R was determined in the presence of competing murine R1 antibody(ML04R1), MAB391, or control non-specific antibody hA20, which binds toCD20. The R1 antibody did not compete for binding to IGF-1R with MAB391.

FIG. 9. Humanized R1 is not an agonist of the IGF-1R receptor. UnlikeIGF-1, hR1 did not stimulate the proliferation of MCF-7 cells inserum-free medium.

FIG. 10. IGF-1R expression in cell lines determined by Guava Expressanalysis using Zenon-labeled antibodies. Expression of IGF-1R wasconfirmed by the binding of hR1 to MCF-7 (breast cancer), CaPan1(pancreatic cancer), and DU-145 (prostate cancer).

FIG. 11. Binding of DNL constructs comprising the hR1 antibody or Fabfragments thereof to cell lines expressing IGF-1R. Hep G2 liver cancercells were incubated with the DNL constructs TF-18 (humanized anti-AFP),1R-31 (humanized anti-AFP/humanized anti-IGF-1R), 1R-15 (humanizedanti-IGF-1R/humanized anti-CEACAM6), 31-1R (humanized anti-AFP IgG andhR1-IgG-AD2).

FIG. 12. Binding of DNL constructs to MCF-7 cells (A), DU-145 cells (B)or ME-180 cells (C), determined on FACScan with DNL constructs or intactantibodies. Hex refers to hexavalent DNL constructs. hRS7 is a humanizedanti-EGP-1 antibody.

FIG. 13. Effect of DNL constructs on neutralizing the growth stimulatingactivity of IGF-1 in DU-145 (A) and ME-180 (B, C) cells expressing bothIGF-1R and EGP-1. The Hex-hR1 construct, comprising anti-IGF-1R,suppressed proliferation of DU-145 (A) and ME-180 (B). The 1R-E1construct comprising anti-IGF-1R and anti-EGP-1, suppressedproliferation of ME-180 (C).

FIG. 14. Down-regulation of IGF-1R in MCF-7 and HT-29 cells treated withhR1, MAB391 and 24-60 antibodies but not hLL2 control antibody.

FIG. 15. Down-regulation of IGF-1R in (A) MCF-7 and DU-145 cells treatedwith Hex-hR1; (B) MCF-7, DU-145 and LNCaP cells treated with Hex-hR1 and1R-E1 DNL constructs.

FIG. 16. Hex-hR1 blocks IGF-1 activation of ERK1/2 phosphorylation inMCF-7 cells. Hex-hR1 and control DNL construct Hex-hRS7 were added at 10nM to cells treated with 100 ng/ml IGF-1.

FIG. 17. 1R-E1, E1-1R and hR1 block IGF-1 activation of IGF-1Rphosphorylation in ME-180 cells. The indicated concentrations of DNLconstruct 1R-E1, E1-R1, hR1 and control hRS7 antibodies were added tocells treated with 100 nM IGF-1

FIG. 18. Hex-hR1 blocks IGF-1 activation of the phosphorylation ofIGF-1R, Akt and ERK1/2 in MCF-7 cells. The indicated concentrations ofDNL construct Hex-hR1, control Hex-hRS7 or hR1 antibody were added tocells treated with 100 ng/ml IGF-1.

FIG. 19. Bispecific hexavalent constructs 1R-E1 or E1-1R inhibitphosphorylation of IGF-1R, Akt and ERK1/2 in MCF-7 cells stimulated with100 ng/ml IGF-1.

FIG. 20. Hex-hR1 inhibits phosphorylation of IGF-1R, Akt and ERK1/2 inDU-145 cells stimulated with 100 ng/ml IGF-1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

As used herein, the term “about” means plus or minus ten percent (10%)of a value. For example, “about 100” would refer to any number between90 and 110.

An antibody refers to a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive, antigen-binding portion of an immunoglobulin molecule, like anantibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-IGF-1R monoclonal antibodyfragment binds to IGF-1R. The term “antibody fragment” also includesisolated fragments consisting of the variable regions, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”). As used herein, the term “antibody fragment” does notinclude portions of antibodies without antigen binding activity, such asFc fragments or single amino acid residues.

A naked antibody or naked antibody fragment refers to an antibody orantigen binding fragment thereof which is not conjugated to atherapeutic agent. Naked antibodies may include murine monoclonalantibodies, as well as recombinant antibodies, such as chimeric,humanized or human antibodies.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,and is useful in the treatment of a disease. Non-limiting examples oftherapeutic agents include antibodies, antibody fragments, drugs,toxins, nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents, oligonucleotides (e.g. anti-senseoligonucleotides or RNAi) and radioisotopes.

A diagnostic agent is a detectable molecule or atom that may beconjugated to an antibody, antibody fragment, targetable construct orother moiety for delivery to a cell, tissue, pathogen or other targetassociated with a disease or medical condition. Useful diagnostic agentsinclude, but are not limited to, radioisotopes, dyes, contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.paramagnetic ions for magnetic resonance imaging). In certainembodiments, a diagnostic agent may be an F-18 labeled moiety (e.g.,U.S. patent application Ser. No. 11/960,262; 12/112,289; PCT PatentApplication Serial No. PCT/US08/62108; the Examples section of each ofwhich is incorporated herein by reference.)

An immunoconjugate is a conjugate of an antibody component with at leastone therapeutic or diagnostic agent. An antibody component may beconjugated with multiple therapeutic and/or diagnostic agents to form animmunoconjugate.

The term antibody fusion protein may refer to a recombinantly producedantigen-binding molecule in which one or more of the same or differentsingle-chain antibody or antibody fragment segments with the same ordifferent specificities are linked. Valency of the fusion proteinindicates how many binding arms or sites the fusion protein has to asingle antigen or epitope; i.e., monovalent, bivalent, trivalent ormultivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindwith one epitope. The fusion protein may comprise a single antibodycomponent, a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators and toxins.One preferred toxin comprises a ribonuclease (RNase), preferably arecombinant RNase. However, the term is not limiting and a variety ofprotein or peptide effectors may be incorporated into a fusion protein.In another non-limiting example, a fusion protein may comprise an AD orDDD sequence for producing a DNL construct as discussed below.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity may be for aB-cell, T-cell, myeloid-, plasma- or mast-cell antigen or epitope.Another specificity may be to a different antigen on the same cell type,such as IGF-1R, CD19, CD20, CD21, CD23, CD45, CD80, HLA-DR, CD74, MUC1,and CD22 on B-cells. However, the second antigen is not limiting andother target antigens of use may be selected from the group consistingof carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3,CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, IGF-1R, CD21,CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L,CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74,CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5,CEACAM6, B7, ED-B fibronectin, Factor H, FHL-1, Flt-3, folate receptor,GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-likegrowth factor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R,IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3,MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, PSMA, EGP-1, EGP-2, AFP, Ia,HM1.24, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAILreceptor (R1 and R2), VEGFR, EGFR, PIGF, complement factors C3, C3a,C3b, C5a, C5, and an oncogene product. Multispecific, multivalentantibodies are constructs that have more than one binding site, and thebinding sites are of different specificity.

In various embodiments, the present invention provides humanized,chimeric or human anti-IGF-1R antibodies, and antibody fusion proteinsthereof, useful for treatment of mammalian subjects, humans and domesticanimals, alone, as a conjugate or administered in combination with othertherapeutic agents, including other naked antibodies and antibodytherapeutic conjugates.

Preferably, the anti-IGF-1R antibody exhibits one or more functionalcharacteristics selected from the group consisting of: (i) binds toIGF-1R but not to IR; (ii) is not an agonist of IGF-1R; (iii) does notblock binding of IGF-1 or IGF-2 to isolated IGF-1R; (iv) effectivelyneutralizes the activation of IGF-1R by IGF-1 in intact cells ortissues; and (v) binds to an epitope of IGF-1R comprising the first halfof the cysteine-rich domain of IGF-1R, between amino acid residues 151and 222 of the human IGF-1R sequence.

In other preferred embodiments, the anti-IGF-1R MAbs or fragmentsthereof comprise the heavy chain variable region complementaritydetermining region (CDR) sequences CDR1 (DYYMY, SEQ ID NO:1), CDR2(YITNYGGSTYYPDTVKG, SEQ ID NO:2) and CDR3 (QSNYDYDGWFAY, SEQ ID NO:3)and the light chain variable region CDR sequences CDR1 (KASQEVGTAVA, SEQID NO:4), CDR2 (WASTRHT, SEQ ID NO:5) and CDR3 (QQYSNYPLT, SEQ ID NO:6).In most preferred embodiments, the anti-IGF-1R antibody or fragmentthereof is hR1.

The humanized anti-IGF-1R MAb or fragment thereof may comprise the CDRsof a murine anti-IGF-1R MAb and the framework (FR) and constant regionsof the light and heavy chain variable regions of one or more humanantibodies, while retaining the IGF-1R targeting specificity of theparent murine anti-IGF-1R MAb. The humanized anti-IGF-1R MAb or fragmentthereof may further comprise at least one amino acid from thecorresponding FRs of the parent murine MAb. The murine framework aminoacid residues can be substituted in the human FR regions of the lightand heavy variable chains if necessary to maintain proper binding or toenhance binding to the IGF-1R antigen. More preferably the humanizedanti-IGF-1R MAb or fragment thereof comprises the amino acid sequencesof hR1VH (SEQ ID NO:9) and hR1VK (SEQ ID NO:10).

Chimeric anti-IGF-1R MAbs or fragments thereof may comprise the variableregion sequences of a murine anti-IGF-1R antibody, attached to humanantibody constant region sequences. In preferred embodiments, thechimeric anti-IGF-1R MAb comprises the heavy and light chain variableregion sequences of murine R1VH (SEQ ID NO:7) and R1VK (SEQ ID NO:8).

Certain embodiments may concern an anti-IGF-1R MAb or fragment thereofthat blocks binding to IGF-1R of a murine, chimeric, humanized or humanantibody comprising the heavy chain complementarity determining region(CDR) sequences CDR1 (DYYMY, SEQ ID NO:1), CDR2 (YITNYGGSTYYPDTVKG, SEQID NO:2) and CDR3 (QSNYDYDGWFAY, SEQ ID NO:3) and the light chain CDRsequences CDR1 (KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQ ID NO:5)and CDR3 (QQYSNYPLT, SEQ ID NO:6).

Other embodiments may encompass antibody fusion proteins comprising atleast one anti-IGF-1R MAb or fragment thereof, as described above. Theantibody fusion protein may comprise at least one first anti-IGF-1R MAbor fragment thereof and at least one second MAb or fragment thereof.More preferably the second MAb binds to an antigen selected from thegroup consisting of B7, CD4, CD5, CD8 CD14, CD15, CD16, CD19, IGF-1R,CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD66a-e, CD70, CD74, CD79a,CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, PAM4 antigen,PSMA, AFP, EGP-1, EGP-2, MIF, ED-B fibronectin, IL-2, IL-6, IL-25, MUC1,MUC2, MUC3, MUC4, MUC5, NCA-90, NCA-95, Ia, HM1.24, HLA-DR, tenascin,T101, TAC, TRAIL-R1, TRAIL-R2, VEGFR, EGFR, PIGF, Flt-3, ILGF,complement factor C5, and an oncogene product. Alternatively the secondMAb may be an anti-IGF-1R MAb that is different than the anti-IGF-1R MAbdescribed herein.

Amino Acid Substitutions

In certain embodiments, the disclosed methods and compositions mayinvolve production and use of antibodies or antigen-binding fragmentsthereof with one or more substituted amino acid residues. As discussedbelow, methods for making monoclonal antibodies against virtually anytarget antigen are well known in the art. Typically, these result inproduction of murine antibodies against a target antigen. As is wellknown in the art, the antigen-binding specificity of murine monoclonalantibodies is determined largely by the hypervariable complementaritydetermining region (CDR) sequences. Murine antibodies generally comprise6 CDR sequences, 3 on the antibody light chain and 3 on the heavy chain.As described in detail below, chimeric, humanized or human versions ofmurine antibodies may be constructed by techniques such as CDR grafting,where the murine CDR sequences are inserted into, for example, humanantibody framework and constant region sequences, or by attaching theentire murine variable region sequences to human antibody constantregion sequences. In alternative embodiments, the variable regionsequences of an antibody may be constructed, for example, by chemicalsynthesis and assembly of oligonucleotides encoding the entire light andheavy chain variable regions of an antibody.

In various embodiments, the structural, physical and/or therapeuticcharacteristics of native, chimeric, humanized or human antibodies maybe optimized by replacing one or more amino acid residues. For example,it is well known in the art that the functional characteristics ofhumanized antibodies may be improved by substituting a limited number ofhuman framework region (FR) amino acids with the corresponding FR aminoacids of the parent murine antibody. This is particularly true when theframework region amino acid residues are in close proximity to the CDRresidues.

In other cases, the therapeutic properties of an antibody, such asbinding affinity for the target antigen, the dissociation- or off-rateof the antibody from its target antigen, or even the effectiveness ofinduction of CDC (complement-dependent cytotoxicity) or ADCC (antibodydependent cellular cytotoxicity) by the antibody, may be optimized by alimited number of amino acid substitutions. Such substitutions may evenoccur, for example, in the CDR portions of the antibody. However, aminoacid substitution is not limited to the CDR or framework regionsequences of antibodies and may also occur, for example, in the Fcportion of an antibody.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5 .+−0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).Replacement of amino acids with others of similar hydrophilicity ispreferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan, tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (O) glu, asn; Glu (E) gln, asp; Gly (G) ala; H is (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For CDR residues, the residue in the free antibody wouldnormally be assumed to be solvent exposed. For interior residues,conservative substitutions would include: Asp and Asn; Ser and Thr; Serand Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu andIle; Leu and Met; Phe and Tyr; Tyr and Trp. (See, e.g., PROWL website atrockefeller.edu) For solvent exposed residues, conservativesubstitutions would include: Asp and Asn; Asp and Glu; Glu and Gln; Gluand Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala andLys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val;Phe and Tyr. (Id.) Various matrices have been constructed to assist inselection of amino acid substitutions, such as the PAM250 scoringmatrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittlematrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Raomatrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,H is, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Preparation of Monoclonal Antibodies Including Chimeric, Humanized andHuman Antibodies

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ), and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999,J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et at, 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity.

A single chain Fv molecule (scFv) comprises a V_(L) domain and a V_(H)domain. The V_(L) and V_(H) domains associate to form a target bindingsite. These two domains are further covalently linked by a peptidelinker (L). Methods for making scFv molecules and designing suitablepeptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80(1995) and R. E. Bird and B. W. Walker, “Single Chain Antibody VariableRegions,” TIBTECH, Vol 9: 132-137 (1991), incorporated herein byreference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an enzymatic cleavage using papain produces twomonovalent Fab fragments and an Fc fragment. These methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein, which patents areincorporated herein in their entireties by reference. Also, see Nisonoffet al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). Pre-targeting methods with bispecific antibodiescomprising at least one binding site for a tumor-associated antigen orother disease target, as well as at one binding site for a targetableconstruct conjugated to therapeutic or diagnostic agents, are also wellknown in the art (see, e.g., U.S. Pat. Nos. 7,300,644; 7,138,103;7,074,405; 7,052,872; 6,962,702; 6,458,933, the Examples section of eachof which is incorporated herein by reference).

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of eachincorporated herein by reference. Reduction of the peptide linker lengthto less than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787 and U.S. PatentApplication Ser. Nos. 11/925,408, filed Oct. 26, 2007, and 12/418,877,filed Apr. 6, 2009; the Examples section of each of which isincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies, either as naked antibody moieties or in combination with awide range of other effector molecules such as immunomodulators,enzymes, chemotherapeutic agents, chemokines, cytokines, diagnosticagents, therapeutic agents, radionuclides, imaging agents,anti-angiogenic agents, growth factors, oligonucleotides, hormones,peptides, toxins, pro-apoptotic agents, or a combination thereof. Any ofthe techniques known in the art for making bispecific or multispecificantibodies may be utilized in the practice of the presently claimedmethods.

Bispecific or multispecific antibodies may incorporate any knownantibody of therapeutic use. Antibodies of use may be commerciallyobtained from a wide variety of known sources. For example, a variety ofantibody secreting hybridoma lines are available from the American TypeCulture Collection (ATCC, Manassas, Va.). A large number of antibodiesagainst various disease targets, including but not limited totumor-associated antigens, have been deposited at the ATCC and/or havepublished variable region sequences and are available for use in theclaimed methods and compositions. See, e.g., U.S. Pat. Nos. 7,312,318;7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060;7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498;7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241;6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107;6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645;6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681;6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405;6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511;6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780;6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711;6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307;6,720,15; 6,716,966; 6,709,653; 6,693,176; 6,692,908; 6,689,607;6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344;6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294;6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572;856;6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625;6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173;6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310; 6,444,206′6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694;6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444;6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459;6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914;6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136;5,587,459; 5,443,953, 5,525,338, the Figures and Examples section ofeach of which is incorporated herein by reference. These are exemplaryonly and a wide variety of other antibodies and their hybridomas areknown in the art. The skilled artisan will realize that antibodysequences or antibody-secreting hybridomas against almost anydisease-associated antigen may be obtained by a simple search of theATCC, NCBI and/or USPTO databases for antibodies against a selecteddisease-associated target of interest. The antigen binding domains ofthe cloned antibodies may be amplified, excised, ligated into anexpression vector, transfected into an adapted host cell and used forprotein production, using standard techniques well known in the art.

Dock-and-Lock (DNL)

In preferred embodiments, bispecific or multispecific antibodies orother constructs may be produced using the dock-and-lock technology(see, e.g., U.S. Pat. Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787and U.S. Patent Application Ser. Nos. 11/925,408, filed Oct. 26, 2007,and 12/418,877, filed Apr. 6, 2009; the Examples section of each ofwhich is incorporated herein by reference). The DNL method exploitsspecific protein/protein interactions that occur between the regulatory(R) subunits of cAMP-dependent protein kinase (PKA) and the anchoringdomain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell. Biol.2004; 5: 959). PKA, which plays a central role in one of the beststudied signal transduction pathways triggered by the binding of thesecond messenger cAMP to the R subunits, was first isolated from rabbitskeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763).The structure of the holoenzyme consists of two catalytic subunits heldin an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RIand RII), and each type has α and β isoforms (Scott, Pharmacol. Ther.1991; 50:123). The R subunits have been isolated only as stable dimersand the dimerization domain has been shown to consist of the first 44amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222).Binding of cAMP to the R subunits leads to the release of activecatalytic subunits for a broad spectrum of serine/threonine kinaseactivities, which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). Interestingly, AKAPs will only bind to dimeric Rsubunits. For human RIIα, the AD binds to a hydrophobic surface formedby the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol.1999; 6:216). Thus, the dimerization domain and AKAP binding domain ofhuman RIIα are both located within the same N-terminal 44 amino acidsequence (Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al.,EMBO J. 2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize DDD (for example, SEQID NO:15 and SEQ ID NO:16) and AD (for example, SEQ ID NO:17 and SEQ IDNO:18) sequences as an excellent pair of linker modules for docking anytwo entities, referred to hereafter as A and B, into a noncovalentcomplex, which could be further locked into a stably tethered structurethrough the introduction of cysteine residues into both the DDD and ADat strategic positions to facilitate the formation of disulfide bonds.The general methodology of the “dock-and-lock” approach is as follows.Entity A is constructed by linking a DDD sequence to a precursor of A,resulting in a first component hereafter referred to as a. Because theDDD sequence would effect the spontaneous formation of a dimer, A wouldthus be composed of a₂. Entity B is constructed by linking an ADsequence to a precursor of B, resulting in a second component hereafterreferred to as b. The dimeric motif of DDD contained in a₂ will create adocking site for binding to the AD sequence contained in b, thusfacilitating a ready association of a₂ and b to form a binary, trimericcomplex composed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chimura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized to link other types of molecules together.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

DNL Sequence Variants

In alternative embodiments, sequence variants of the AD and/or DDDmoieties may be utilized in construction of the DNL complexes. Thestructure-function relationships of the AD and DDD domains have been thesubject of investigation. (See, e.g., Burns-Hamuro et al., 2005, ProteinSci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto etal., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006,Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Goldet al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:15below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

Human DDD Sequence from Protein Kinase A

(SEQ ID NO: 15) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:17), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:17.

AKAP-IS SEQUENCE QIEYLAKQIVDNAIQQA (SEQ ID NO: 17)

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:39), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:40-42. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AKAP-IS sequence (SEQ ID NO:17), theAD moiety may also include the additional N-terminal residues cysteineand glycine and C-terminal residues glycine and cysteine, as shown inSEQ ID NO:18.

SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 39) Alternative AKAPsequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 40) QIEYHAKQIVDHAIHQA (SEQ IDNO: 41) QIEYVAKQIVDHAIHQA (SEQ ID NO: 42)

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:43-45. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:43), RIAD (SEQ ID NO:44) and PV-38 (SEQ IDNO:45). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 43) RIAD LEQYANQLADQIIKEATE(SEQ ID NO: 44) PV-38 FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 45)

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence. The residues are the same asobserved by Alto et al. (2003), with the addition of the C-terminalalanine residue. (See FIG. 4 of Hundsrucker et al. (2006), incorporatedherein by reference.) The sequences of peptide antagonists withparticularly high affinities for the RII DDD sequence are shown in SEQID NO:46-48.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 17) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 46) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 47) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 48)

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:15. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins.

(SEQ ID NO: 15) SHIQ IP P GL TE LL Q G Y T V EVLR Q QP P DL VE FA VE YFTR L REA R A

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

scFv-Based AD Modules

Alternative embodiments may concern the use of scFV-based AD modules forpairing with DDD2 (SEQ ID NO:16) based cytokines or RNase to yield DNLconjugates that are smaller. The smaller sized DNL constructs mayfacilitate penetration into solid tumors. We have produced several typesof scFv-based bispecific antibodies by expressing two discretepolypeptide chains comprising complementary variable domains with a6-His tag (SEQ ID NO: 51) at the carboxyl terminus of each polypeptidechain. The same approach may be used to generate scFv-based AD modulesby replacing one or both 6-His tags (SEQ ID NO: 51) with either an ADsequence or an AD-HHHHHH sequence (SEQ ID NO: 51). We can also fuse eachpolypeptide chain with a different AD sequence (e.g. AD2 (SEQ ID NO:18)and AD3 (SEQ ID NO:49)), which would allow the specific recognition byits cognate DDD sequence, thus providing further complexicity of thefinal DNL conjugates. Table 1 below provides a non-exhaustive list ofsuch scFv-based DNL constructs.

TABLE 1 scFv-based DNL constructs Configuration ScFv-AD MW (kDa) NoteBS2 I VH₁-VL₂-AD2 30 Bispecific, VH₂-VL₁ 25 1 × 1 II VH₁-VL₂-AD2 30VH₂-VL₁-AD2 30 III VH₁-VL₂-AD2 30 VH₂-VL₁-AD3 30 “DVD” I VH₁-VH₂-AD2 30Bispecific, VL₁-VL₂ 25 1 × 1 II VH₁-VH₂-AD2 30 VL₂-VL₁-AD2 30 IIIVH₁-VH₂-AD2 30 VL₂-VL₁-AD3 30 BS6 I VH₁-VL₁-VH₂-AD2 45 Bispecific,VL₂-VH₁-VL₁ 40 2 × 1 II VH₁-VL₁-VH₂-AD2 45 VL₂-VH₁-VL₁-AD2 45 IIIVH₁-VL₁-VH₂-AD2 45 VL₂-VH₁-VL₁-AD3 BS8 I VH₁-VH₁-VH₂-AD2 45 Bispecific,VL₂-VL₁-VL₁ 40 2 × 1 II VH₁-VH₁-VH₂-AD2 45 VL₂-VL₁-VL₁-AD2 45 IIIVH₁-VH₁-VH₂-AD2 45 VL₂-VL₁-VL₁-AD3 45 BS18 I VH₁-CH₁-VH₂-AD2 55VL₁-CL-VL₂ 50 II VH₁-CH₁-VH₂-AD2 55 VL₁-CL-VL₂-AD2 55 IIIVH₁-CH₁-VH₂-AD2 55 VL₁-CL-VL₂-AD3 55 TS I VH₁-VH₂-VH₃-AD2 45Trispecific, VL₃-VL₂-VL₁ 40 1 × 1 × 1 II VH₁-VH₂-VH₃-AD2 45VL₃-VL₂-VL₁-AD2 45 III VH₁-VH₂-VH₃-AD2 45 VL₃-VL₂-VL₁-AD3 45

Type I is designed to link one pair of DDD2 (SEQ ID NO:16) modules. TypeII is designed to link two pairs of the same or different DDD2 modules.Type III is designed to link one pair of DDD2 modules and one pair ofDDD3 (SEQ ID NO:50) modules. The two polypeptides chains are designed toassociate in an anti-parallel fashion.

Pre-Targeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct ortargetable construct) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702;6,962,702; 7,074,405; and U.S. Ser. No. 10/114,315 (now abandoned); theExamples section of each of which is incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents. The technique mayalso be utilized for antibody dependent enzyme prodrug therapy (ADEPT)by administering an enzyme conjugated to a targetable construct,followed by a prodrug that is converted into active form by the enzyme.

Avimers

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220.) The resultingmultidomain proteins may comprise multiple independent binding domains,that may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDD and/orAD sequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756 (now abandoned), 20050048512 (now abandoned), 20050053973(now abandoned), 20050089932 and 20050221384 (now abandoned), theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods forpreparing a phage library. The phage display technique involvesgenetically manipulating bacteriophage so that small peptides can beexpressed on their surface (Smith and Scott, 1985, Science228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257). Inaddition to peptides, larger protein domains such as single-chainantibodies may also be displayed on the surface of phage particles (Arapet al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of palming. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, theExamples section of each incorporated herein by reference. Methods forpreparation and screening of aptamers that bind to particular targets ofinterest are well known, for example U.S. Pat. No. 5,475,096 and U.S.Pat. No. 5,270,163, the Examples section of each incorporated herein byreference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR', CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S, Not all linkages in an oligomer need to beidentical.

Therapeutic and Diagnostic Agents

In certain embodiments, the antibodies, antibody fragments or fusionproteins described herein may be administered alone, as a “naked”antibody, fragment or fusion protein. In alternative embodiments, theantibody, fragment or fusion protein may be administered either before,concurrently with, or after at least one other therapeutic agent. Inother alternatives, an antibody, fragment or fusion protein may becovalently or non-covalently attached to at least one therapeutic and/ordiagnostic agent to form an immunoconjugate.

Therapeutic agent are preferably selected from the group consisting of aradionuclide, an immunomodulator, an anti-angiogenic agent, a cytokine,a chemokine, a growth factor, a hormone, a drug, a prodrug, an enzyme,an oligonucleotide, a pro-apoptotic agent, an interference RNA, aphotoactive therapeutic agent, a cytotoxic agent, which may be achemotherapeutic agent or a toxin, and a combination thereof. The drugsof use may possess a pharmaceutical property selected from the groupconsisting of antimitotic, antikinase, alkylating, antimetabolite,antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents andcombinations thereof.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,aplidin, azaribine, anastrozole, anthracyclines, bendamustine,bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide,estramustine, epidophyllotoxin, estrogen receptor binding agents,etoposide (VP 16), etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide,leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, navelbine, nitrosurea, plicomycin, procarbazine, paclitaxel,pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen,taxol, temazolomide (an aqueous form of DTIC), transplatinum,thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracilmustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, an hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

Radioactive isotopes useful for treating diseased tissue include, butare not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, , ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho,¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo. ¹⁰⁵Rh,¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, and ²¹¹Pb. The therapeuticradionuclide preferably has a decay-energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV. Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe,⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like. Some useful diagnosticnuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Cu, ⁶⁷Ga, ⁸⁶Y, ⁸⁹Zr,⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or ¹¹¹In. In certain embodiments, anti-IGF-1Rantibodies, such as hR1, may be of use in combination with therapeuticradionuclides for sensitization of tumors to radiation therapy (see,e.g., Allen et al., 2007, Cancer Res. 67:1155).

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Jori et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Corticosteroid hormones can increase the effectiveness of otherchemotherapy agents, and consequently, they are frequently used incombination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-placenta growth factor (PIGF)peptides and antibodies, anti-vascular growth factor antibodies (such asanti-VEGF and anti-PIGF), anti-Flk-1 antibodies, anti-Flt-1 antibodiesand peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF(macrophage migration-inhibitory factor) antibodies, laminin peptides,fibronectin peptides, plasminogen activator inhibitors, tissuemetalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-β,thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16Kprolactin fragment, Linomide, thalidomide, pentoxifylline, genistein,TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir,vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline maybe of use.

Other useful therapeutic agents comprise oligonucleotides, especiallyantisense oligonucleotides that preferably are directed againstoncogenes and oncogene products, such as bcl-2.

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹¹⁰In,¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III). Ultrasound contrast agents may comprise liposomes, suchas gas filled liposomes. Radiopaque diagnostic agents may be selectedfrom compounds, barium compounds, gallium compounds, and thalliumcompounds. A wide variety of fluorescent labels are known in the art,including but not limited to fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. Chemiluminescent labels of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt or an oxalate ester.

Immunoconjugates

Any of the antibodies, antibody fragments or antibody fusion proteinsdescribed herein may be conjugated to one or more therapeutic ordiagnostic agents. The therapeutic agents do not need to be the same butcan be different, e.g. a drug and a radioisotope. For example, ¹³¹I canbe incorporated into a tyrosine of an antibody or fusion protein and adrug attached to an epsilon amino group of a lysine residue. Therapeuticand diagnostic agents also can be attached, for example to reduced SHgroups and/or to carbohydrate side chains. Many methods for makingcovalent or non-covalent conjugates of therapeutic or diagnostic agentswith antibodies or fusion proteins are known in the art and any suchknown method may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody componenthaving an oxidized carbohydrate portion with a carrier polymer that hasat least one free amine function. This reaction results in an initialSchiff base (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos.5,443,953 and 6,254,868, the Examples section of which is incorporatedherein by reference. The engineered carbohydrate moiety is used toattach the therapeutic or diagnostic agent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein or to a targetable construct andused to chelate a therapeutic or diagnostic agent, such as aradionuclide. Exemplary chelators include but are not limited to DTPA(such as Mx-DTPA), DOTA, TETA, NETA or NOTA. Methods of conjugation anduse of chelating agents to attach metals or other ligands to proteinsare well known in the art (see, e.g., U.S. Patent Application No.7,563,433, the Examples section of which is incorporated herein byreference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MRI. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F—Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats, comprising administering to the subject a therapeuticallyeffective amount of an antibody, fragment or fusion protein. Inpreferred embodiments, the antibody or fragment is an anti-IGF-1R MAb.In certain embodiments, the therapy may utilize a “naked antibody” thatdoes not have a therapeutic agent bound to it.

The administration of a “naked” anti-IGF-1R antibody can be supplementedby administering concurrently or sequentially a therapeuticallyeffective amount of another “naked antibody” that binds to or isreactive with another antigen on the surface of the target cell.Preferred additional MAbs comprise at least one humanized, chimeric orhuman MAb selected from the group consisting of a MAb reactive with CD4,CD5, CD8, CD14, CD15, CD16, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25,CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54,CD70, CD74, CD79a, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5,CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase IX, PAM4antigen, MUC1, MUC2, MUC3, MUC4, MUC5, Ia, MIF, HM1.24, HLA-DR,tenascin, Flt-3, VEGFR, PIGF, ILGF, IL-6, IL-25, tenascin, TRAIL-R1,TRAIL-R2, complement factor C5, oncogene product, or a combinationthereof.

The naked anti-IGF-1R therapy alone or in combination with other nakedMAbs can be further supplemented with the administration, eitherconcurrently or sequentially, of at least one therapeutic agent, asdiscussed above. Multimodal therapies may include therapy with nakedanti-IGF-1R antibodies supplemented with administration of anti-CD22,anti-CD19, anti-CD20, anti-CD21, anti-CD74, anti-CD80, anti-CD23,anti-CD45, anti-CD46, anti-MIF, anti-EGP-1, anti-CEACAM5, anti-CEACAM6,PAM4, or anti-HLA-DR (including the invariant chain) antibodies in theform of naked antibodies, fusion proteins, or as immunoconjugates. Thenaked anti-IGF-1R antibodies or fragments thereof may also besupplemented with naked antibodies against a MUC1 or MUC5 antigen.Various antibodies of use, such as anti-CD19, anti-CD20, and anti-CD22antibodies, are known to those of skill in the art. See, for example,Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., CancerImmunol. Immunother. 32:364 (1991); Longo, Curr. Opin. Oncol. 8:353(1996), U.S. Pat. Nos. 5,798,554; 6,187,287; 6,306,393; 6,676,924;7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785; 7,238,786;7,282,567; 7,300,655; 7,312,318; 7,612,180; 7,501,498 and U.S. PatentApplication Publ. Nos. 20080131363; 20080089838; 20070172920;20060193865; and 20080138333; the Examples section of each of which isincorporated herein by reference.

In another form of multimodal therapy, subjects receive nakedanti-IGF-1R antibodies, and/or immunoconjugates, in conjunction withstandard cancer chemotherapy. For example, “CVB” (1.5 g/m²cyclophosphamide, 200-400 mg/m² etoposide, and 150-200 mg/m² carmustine)is a regimen used to treat non-Hodgkin's lymphoma. Patti et al., Eur. J.Haematol. 51: 18 (1993). Other suitable combination chemotherapeuticregimens are well-known to those of skill in the art. See, for example,Freedman et al., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME2, 3rd Edition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger1993). As an illustration, first generation chemotherapeutic regimensfor treatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate, bendamustine, and bryostatin-1. In a preferredmultimodal therapy, both chemotherapeutic drugs and cytokines areco-administered with an antibody, immunoconjugate or fusion proteinaccording to the present invention. The cytokines, chemotherapeuticdrugs and antibody or immunoconjugate can be administered in any order,or together.

Immunoconjugates or naked antibodies can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe immunoconjugate or naked antibody is combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON′SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof

The immunoconjugate or naked antibody of the present invention can beformulated for intravenous administration via, for example, bolusinjection or continuous infusion. Preferably, the antibody of thepresent invention is infused over a period of less than about 4 hours,and more preferably, over a period of less than about 3 hours. Forexample, the first 25-50 mg could be infused within 30 minutes,preferably even 15 min, and the remainder infused over the next 2-3 hrs.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et at,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON′S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate, antibody fusion proteins, or naked antibody mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. Preferably, the antibody isinfused over a period of less than about 4 hours, and more preferably,over a period of less than about 3 hours.

More generally, the dosage of an administered immunoconjugate, fusionprotein or naked antibody for humans will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. It may be desirable to providethe recipient with a dosage of immunoconjugate, antibody fusion proteinor naked antibody that is in the range of from about 1 mg/kg to 25 mg/kgas a single intravenous infusion, although a lower or higher dosage alsomay be administered as circumstances dictate. A dosage of 1-20 mg/kg fora 70 kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a1.7-m patient. The dosage may be repeated as needed, for example, onceper week for 4-10 weeks, once per week for 8 weeks, or once per week for4 weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy.

Alternatively, an antibody such as a naked anti-IGF-1R, may beadministered as one dosage every 2 or 3 weeks, repeated for a total ofat least 3 dosages. Or, the antibodies may be administered twice perweek for 4-6 weeks. If the dosage is lowered to approximately 200-300mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kgpatient), it may be administered once or even twice weekly for 4 to 10weeks. Alternatively, the dosage schedule may be decreased, namely every2 or 3 weeks for 2-3 months. It has been determined, however, that evenhigher doses, such as 20 mg/kg once weekly or once every 2-3 weeks canbe administered by slow i.v. infusion, for repeated dosing cycles. Thedosing schedule can optionally be repeated at other intervals and dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the anti-IGF-1R antibodies are of use fortherapy of cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor). Cancers conducive to treatment methods of thepresent invention involves cells which express, over-express, orabnormally express IGF-1R.

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).Such conditions in which cells begin to express, over-express, orabnormally express IGF-1R, are particularly treatable by the disclosedmethods and compositions.

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one antibody, fragment or fusion protein as describedherein. If the composition containing components for administration isnot formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an antibody, fragment, fusion protein or bispecificantibody. Exemplary sequences that may be encoded and expressed includean anti-IGF-1R MAb or fragment thereof, a fusion protein comprising atleast one anti-IGF-1R antibody or fragment thereof, a fusion proteincomprising at least one first antibody or fragment and at least onesecond antibody or fragment. The first and second antibodies maycomprise an anti-IGF-1R antibody, an antibody against a tumor associatedantigen and/or a hapten on a targetable construct. Fusion proteins maycomprise an antibody or antibody fragment attached to a differentpeptide or protein, such as the AD and DDD peptides utilized for DNLconstruct formation as discussed in more detail in the Examples below.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express MAbs in a selected host cell, immunoglobulinenhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci. USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence asdiscussed below.

Also encompassed is a method of expressing antibodies or fragmentsthereof or fusion proteins. The skilled artisan will realize thatmethods of genetically engineering expression constructs and insertioninto host cells to express engineered proteins are well known in the artand a matter of routine experimentation. Host cells and methods ofexpression of cloned antibodies or fragments have been described, forexample, in U.S. Pat. Nos. 7,531,327; 7,537,930; and 7,608,425, theExamples section of each of which is incorporated herein by reference.

General Techniques for Construction of Anti-IGF-1R Antibodies

The V_(κ) (variable light chain) and V_(H) (variable heavy chain)sequences for anti-IGF-1R antibodies may be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. The V genes of an anti-IGF-1R MAb from a cell that expressesa murine anti-IGF-1R MAb can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized anti-IGF-1R MAb can then be designedand constructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine anti-IGF-1R MAb by general molecular cloningtechniques (Sambrook et al., Molecular Cloning, A laboratory manual,2^(nd) Ed (1989)). The V_(κ) sequence for the MAb may be amplified usingthe primers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extendedprimer set described by Leung et al. (BioTechniques, 15: 286 (1993)).The V_(H) sequences can be amplified using the primer pairVH1BACK/VH1FOR (Orlandi et al., 1989) or the primers annealing to theconstant region of murine IgG described by Leung et al. (Hybridoma,13:469 (1994)).

PCR reaction mixtures containing 10 μl of the first strand cDNA product,10 μl of 10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mMMgCl₂, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.),250 μM of each dNTP, 200 nM of the primers, and 5 units of Taq DNApolymerase (Perkin Elmer Cetus) can be subjected to 30 cycles of PCR.Each PCR cycle preferably consists of denaturation at 94° C. for 1 min,annealing at 50° C. for 1.5 min, and polymerization at 72° C. for 1.5min. Amplified V_(κ) and VH fragments can be purified on 2% agarose(BioRad, Richmond, Calif.). The humanized V genes can be constructed bya combination of long oligonucleotide template syntheses and PCRamplification as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

PCR products for V_(κ) can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the V_(κ)PCR products. PCR products for V_(H) canbe subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Individual clones containing the respective PCRproducts may be sequenced by, for example, the method of Sanger et al.(Proc. Natl. Acad. Sci., USA, 74: 5463 (1977)).

Expression cassettes containing the V_(κ) and V_(H) sequences, togetherwith the promoter and signal peptide sequences, can be excised fromVKpBR and VHpBS, respectively, by double restriction digestion asHindIII-BamHI fragments. The V_(κ) and V_(H) expression cassettes can beligated into appropriate expression vectors, such as pKh and pG1g,respectively (Leung et al., Hybridoma, 13:469 (1994)). The expressionvectors can be co-transfected into an appropriate cell, e.g., myelomaSp2/0-Ag14 (ATCC, VA), colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of a chimeric, humanized orhuman anti-IGF-1R MAb by, for example, an ELISA assay. Alternatively,the V_(κ) and V_(H) expression cassettes can be assembled in themodified staging vectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI andXhoI/BamHI fragments, respectively, and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)). Another vector that is useful is the GS vector, asdescribed in Barnes et al., Cytotechnology 32:109-123 (2000). Otherappropriate mammalian expression systems are described in Werner et al.,Arzneim.-Forsch./Drug Res. 48(II), Nr. 8, 870-880 (1998).

Co-transfection and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of VKpKh (light chain expressionvector) and 20 μg of VHpG1g (heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992). Following transfection, cells may be grown in 96-wellmicrotiter plates in complete HSFM medium (Life Technologies, Inc.,Grand Island, N.Y.) at 37° C., 5% CO₂. The selection process can beinitiated after two days by the addition of hygromycin selection medium(Calbiochem, San Diego, Calif.) at a final concentration of 500 units/mlof hygromycin. Colonies typically emerge 2-3 weeks post-electroporation.The cultures can then be expanded for further analysis. Transfectomaclones that are positive for the secretion of chimeric, humanized orhuman heavy chain can be identified by ELISA assay.

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M citrate buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by absorbance at 280 nm and its concentration adjusted toabout 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is convenientlyadded to the sample as preservative.

EXAMPLES Example 1 Generation and Initial Characterization ofAnti-IGF-1R Antibodies: R1, cR1, and hR1

Three BALB/c mice were each immunized i.p. with 15 μg of recombinanthuman IGF-1R (R&D Systems, Catalog #391-GR), comprising a mixture ofboth processed and unprocessed extracellular domain of human IGF-1R, incomplete Freund's adjuvant. Additional immunizations in incompleteFreund's adjuvant were done 14, 21, and 28 days after the initialimmunization. Spleen cells from the immunized mice were fused withP3X63Ag8.653 cells to generate hybridomas according to standardprotocols. One clone (C-11) expressing anti-IGF-1R but not anti-IR(insulin receptor) activity was isolated and expanded in cultures toobtain the mouse antibody designated ML04R1 or R1, which was shown to bean IgG1/k with the ability to inhibit the binding of radioiodinatedIGF-1 to the IGF-1R expressing human breast cancer cell line MCF-7L (asubline of MCF-7) comparable to a commercially available mouseanti-IGF-1R monoclonal antibody (mAb) MAB391 (Table 2).

TABLE 2 Binding of ¹²⁵I-IGF-1 to MCF-7L in the presence of MAB391 or R1[Ab] MAB391^(a) R1 1000 ng/mL  38%  58% 100 ng/mL  54%  71% 10 ng/mL 95%  97% 0 ng/mL 100% 100% ^(a)R&D clone 33255.111

To obtain cR1, the mouse-human chimeric mAb of R1, the V_(H) and V_(K)genes of R1 were cloned by 5′-RACE. The authenticity of the cloned V_(H)and V_(K) genes was confirmed by N-terminal protein sequencing thatshowed an exact match of the first 15 N-terminal amino acids with thecorresponding amino acids deduced from DNA sequences (Table 3). Thecloned V_(H) and V_(K) genes were inserted into the pdHL2 vector togenerate cRlpdHL2 (FIG. 1), the expression vector for cR1.

TABLE 3 N-terminal protein sequencing of R1 Cycle position 1 2 3 4 5 6 78 9 10 11 12 13 14 15 (H + L)^(a) D/E I/V K/V L/M T/V E/Q S G/H G/K F/GL/M S/V Q/T P/S G/V V_(H) ^(b) (SEQ ID E V K L V E S G G G L V Q P G NO:52) V_(K) ^(b) (SEQ ID D I V M T Q S H K F M S T S V NO: 53)^(a)Purified R1 was subjected to N-terminal protein sequencing (15cycles). Two residues were detected after each cycle of Edmandegradation. ^(b)Deduced from the DNA sequences.

cR1-producing clones were generated using SpE-26 (e.g., U.S. Pat. No.7,531,327), a variant of Sp2/0-Ag14 that shows improved growthproperties, as host cells. Briefly, approximately 30 μg of cRlpdHL2 waslinearized by digestion with Sail restriction endonuclease andtransfected into SpE-26 cells by electroporation. The transfectants wereselected with 0.075 μM methotrexate (MTX), and screened by ELISA forhuman Fc binding activities. The higher producing clones were furtherexpanded to pick the two best clones (709.2D2 and 710.2G2), from whichcR1 was produced in batch cultures, purified by Protein A, and eachconfirmed by ELISA to bind specifically to immobilized rhIGF-1R, but notto immobilized rhIR, as shown in FIG. 2, with the same high affinity(K_(D)˜0.1 nM) for immobilized rhIGF-1R, as shown in FIG. 3.Surprisingly, cR1 appears to have a higher affinity than R1 for rhIGF-1Rimmobilized onto polystyrene beads as shown by a competition assay inwhich the binding of R1 tagged with a fluorescent probe was measured byflow cytometry in the presence of varying concentrations of cR1 or R1(FIG. 4).

Successful humanization of cR1 to hR1 was achieved by grafting the CDRsonto the human framework regions of hMN-14 (U.S. Pat. Nos. 5,874,540 and6,676,924, the Examples section of each incorporated herein byreference) in which certain human framework residues were replaced withmurine counterparts of R1 at corresponding positions. Other selectedresidues were substituted for cloning purposes, resulting in the aminoacid sequences of hR1V_(H) and hR1V_(K) as shown in SEQ ID NO:9 and SEQID NO:10, respectively. Genes encoding hR1 V_(H) and hR1Vk were thensynthesized and engineered into pdHL2 to obtain hR1pdHL2, the expressionvector for hR1. Subsequent efforts to secure the production clone(711.3C11) for hR1 were similar to those describe above for cR1.Positive clones were selected for binding activity to rhIGF-1R. As shownin FIG. 5, hR1 displayed virtually the same binding affinity as cR1 forrhIGF-1R immobilized on polystyrene beads.

To determine whether cR1 can block the binding of IGF-1 or IGF-2 toIGF-1R, we used polystyrene beads immobilized with rhIGF-1R assurrogates of cells expressing IGF-1R and performed thebeads-competition assays as follows. Briefly, varying concentrations (0to 670 nM) of cR1, IGF-1, or IGF-2 were mixed with a constant amount of¹²⁵I-IGF-1 or ¹²⁵I-IGF-2. The rhIGF-1-coated beads were then added,incubated at room temperature for 1 h with gentle rocking, washed, andcounted for radioactivity. The results shown in FIG. 6 indicate that cR1failed to block the binding of either IGF-1 or IGF-2 to such immobilizedrhIGF-1R under these conditions. The results of a similar experimentshown in FIG. 7 also indicate that binding of ¹²⁵I -IGF-1 to thebead-immobilized IGF-1R was effectively blocked by IGF-1 or MAB391, butnot by hR1 or R1. These findings suggest IGF-1 and MAB391 bind to thesame epitope, or have overlapping epitopes of IGF-1R, and hR1 targets adifferent region of IGF-1R from MAB391 or IGF-1. As the primary bindingsite of IGF-1R for IGF-1 was reported to be located in the cysteine-rich(CR) domain between amino acids (aa) 223 and 274, and the same region(aa 223-274) has been assigned as the epitope to αIR-3, which likeMAB391, competes for IGF-1 binding (Gustafson T A, Rutter W J. J BiolChem 1990; 265:18663-7), it appeared that MAB391 also binds to the sameregion or interacts with sites in close proximity.

Example 2 Epitope Mapping Studies of R1, cR1, and hR1

To further locate the region of IGF-1R to which hR1 binds, a panel ofcommercially available anti-IGF-1R mAbs that have their epitopes toIGF-1R mapped, were evaluated for their ability to cross-block eachother from binding to the IGF-1R-coated beads. The results of twotypical experiments are provided in FIG. 8A, which shows the binding ofR1 tagged with a fluorescent probe (PE) was not affected by MAB391 evenat 100 μg/mL, and FIG. 8B, which shows the binding of MAB391 tagged withPE was only partially inhibited (50 to 60%) by R1 at 100 μg/mL.Additional results summarized in Table 3A indicate that the epitope ofR1 is located in the CR domain between aa 151 and 282 and can be furtherlocated to the first half of the CR domain between aa 151 and 222 (Table3B).

TABLE 3A % binding of each labeled antibody (*) to rhIGF-1R-coated beadsin the presence of the unlabeled antibody (24-31, 24-57, 17-69, 1-2,1H7, 2C8, 3B7) Anti-IGF-1R 24-31 24-57 17-69 1-2 1H7 2C8 3B7 Epitope283-440 440-514 514-586 1323-1337 ? (301-450?) (1-150?) R1* 100 100 100100 150 100 117 cR1* 100 100 100 100 125 100 106 hR1* 100 100 100 100131 100 100 MAB391* ND ND ND ND ND ND ND 24-60* 18 88 82 100 100 88 79αIR-3* 52 87 89 95 115 97 76

TABLE 3B % binding of each labeled antibody (*) to rhIGF-1R-coated beadsin the presence of the unlabeled antibody (R1, cR1, hR1, 24-60, αIR-3,MAB391) Anti-IGF-1R R1 cR1 hR1 24-60 αIR-3 MAB391 Epitope (151-282)(151-282) (151-282) 184-283 223-274 (184-283) 151-222 R1* 0 0 0 43 143100 cR1* 0 0 0 40 128 108 hR1* 0 0 0 71 136 121 24-60* 0 0 0 0 21 0αIR-3* 86 97 107 0 0 0 MAB391* 40 ND ND ND ND 0

Example 3 Additional Characterization of R1, cR1, and hR1

Whereas IGF-1 stimulates proliferation of MCF-7 cells grown inserum-free medium, achieving a maximal effect of 50% increase in viablecell counts at 100 ng/mL when compared to the untreated control at 48 h,hR1 does not (FIG. 9). Thus hR1 is not agonistic upon binding to IGF-1R.Internalization of hR1 into MCF-7 was observed at 37° C. but not at 4°C. (not shown).

Example 4 Construction of Expression Vectors for hR1-IgG4(S228P) Variant

B13-24 cells containing an IgG4 gene are purchased from ATCC (ATCCNumber CRL-11397) and genomic DNA is isolated. Briefly, cells are washedwith PBS, resuspended in digestion buffer (100 mM NaCl, 10 mM Tris-HClpH8.0, 25 mM EDTA pH8.0, 0.5% SDS, 0.1 mg/ml proteinase K) and incubatedat 50° C. for 18 h. The sample is extracted with an equal volume ofphenol/chloroform/isoamylalcohol and precipitated with 7.5 MNH.sub.4Ac/100% EtOH. Genomic DNA is recovered by centrifugation anddissolved in TE buffer. Using genomic DNA as template, the IgG4 gene isamplified by PCR using the following primers.

Primer-SacII (SEQ ID NO: 11)CCGCGGTCACATGGCACCACCTCTCTTGCAGCTTCCACCAAGGGCCC Primer-EagI: (SEQ ID NO:12) CCGGCCGTCGCACTCATTTACCCAGAGACAGGG

Amplified PCR product is cloned into a TOPO-TA sequencing vector(Invitrogen) and confirmed by DNA sequencing. The SacII-EagI fragmentcontaining the heavy chain constant region of IgG1 in hR1pdHL2 isreplaced with SacII-EagI of the TOPO-TA-IgG4 plasmid to produce thehR1-pdHL2-IgG4 (hR1pdHL2-γ4) vector.

IgG4-Proline Mutation

A Ser228Pro mutation is introduced in the hinge region of IgG4 to avoidformation of half-molecules. A mutated hinge region 56 by fragment(PstI-StuI) is synthesized

Top (SEQ ID NO: 13) GAGTCCAAATATGGTCCCCCATGCCCACCGTGCCCAGGTAAGCCAACCCAGG; Bottom: (SEQ ID NO: 14)CCTGGGTTGGCTTACCTGGGCACGGTGGGCATGGGGGACCATATTTGGAC TCTGCAannealed and replaced with the PstI-StuI fragment of IgG4. Thisconstruction results in a final vector hR1pdHL2-γ4P.

Example 5 Generation of Multivalent hR1-based Antibodies by DNL

The DNL technique may be used to make multivalent, hR1-based antibodiesin various formats that are either monospecific or bispecific. Forcertain preferred embodiments, Fab antibody fragments may be produced asfusion proteins containing either a DDD or AD sequence. Bispecificantibodies may be formed by combining a Fab-DDD fusion protein of afirst antibody with a Fab-AD fusion protein of a second antibody.Alternatively, an IgG-AD module may be produced as a fusion protein andcombines with a Fab-DDD module of the same or different specificity.Additional types of constructs may be made that combine the targetingcapabilities of an antibody with the effector function of any otherprotein or peptide.

Independent transgenic cell lines are developed for each DDD- orAD-fusion protein. Once produced, the modules can be purified if desiredor maintained in the cell culture supernatant fluid. Followingproduction, any Fab-DDD module can be combined with any AD-module. DDD-or AD-modules may be produced synthetically such as linkinging anAD-sequence to polyethylene glycol or a DDD-sequence to anoligonucleotide. For different types of constructs, different AD or DDDsequences may be utilized.

(SEQ ID NO: 15) DDD1: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQID NO: 16) DDD2: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ IDNO: 17) AD1: QIEYLAKQIVDNAIQQA (SEQ ID NO: 18) AD2:CGQIEYLAKQIVDNAIQQAGC

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See, Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD, Fab-AD, or IgG-ADexpression vectors, as described in detail below for Fab-DDD1 andFab-AD1. To generate the expression vector for Fab-DDD1, the codingsequences for the hinge, C_(H)2 and C_(H)3 domains of the heavy chainare replaced with a sequence encoding the first 4 residues of the hinge,a 14 residue Gly-Ser linker and DDD1 (the first 44 residues of humanRIIα). To generate the expression vector for Fab-AD1, the sequences forthe hinge, C_(H)2 and C_(H)3 domains of IgG are replaced with a sequenceencoding the first 4 residues of the hinge, a 15 residue Gly-Ser linkerand AD1 (a 17 residue synthetic AD called AKAP-IS, which was generatedusing bioinformatics and peptide array technology and shown to bind RIIαdimers with a very high affinity (0.4 nM). See Alto, et al. Proc. Natl.Acad. Sci., U.S.A (2003), 100:4445-50).

To facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD1 orFab-AD1 expression vectors, two shuttle vectors were designed andconstructed as follows.

Preparation of C_(H)1

The C_(H)1 domain was amplified by PCR using the pdHL2 plasmid vector asa template. The left PCR primer consists of the upstream (5′) end of theC_(H)1 domain and a SacII restriction endonuclease site, which is 5′ ofthe C_(H)1 coding sequence. The right primer consists of the sequencecoding for the first 4 residues of the hinge (PKSC) followed by fourglycines and a serine, with the final two codons (GS) comprising a BarnHI restriction site.

5′ of C_(H)l Left Primer (SEQ ID NO: 19) 5′GAACCTCGCGGACAGTTAAG-3′C_(H)l + G₄S-Bam Right (“G₄S” disclosed as SEQ ID NO: 54) (SEQ ID NO:20) 5′GGATCCTCCGCCGCCGCAGCTCTTAGGTTTCTTGTCCACCTTGGTGTT GCTGG-3′

The 410 by PCR amplimer was cloned into the pGemT PCR cloning vector(Promega, Inc.) and clones were screened for inserts in the T7 (5′)orientation.

Construction of (G₄S)₂DDD1 (“(G₄S)₂” disclosed as SEQ ID NO: 55)

A duplex oligonucleotide, designated (G₄S)₂DDD1 (“(G₄S)₂″ disclosed asSEQ ID NO: 55), was synthesized by Sigma Genosys (Haverhill, UK) to codefor the amino acid sequence of DDD1 preceded by 11 residues of thelinker peptide, with the first two codons comprising a BamHI restrictionsite. A stop codon and an EagI restriction site are appended to the 3′end. The encoded polypeptide sequence is shown below.

(SEQ ID NO: 21) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA

The two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom,that overlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGenosys) and combined to comprise the central 154 base pairs of the 174by DDD1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase.

RIIA1-44 top (SEQ ID NO: 34)5′GTGGCGGGTCTGGCGGAGGTGGCAGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAGGTGCTGCGACAG-3′ RIIA1-44 bottom (SEQ IDNO: 35) 5′GCGCGAGCTTCTCTCAGGCGGGTGAAGTACTCCACTGCGAATTCGACGAGGTCAGGCGGCTGCTGTCGCAGCACCTCCACCGTGTAGCCCTG-3′

Following primer extension, the duplex was amplified by PCR using thefollowing primers:

G4S Bam-Left (“G4S” disclosed as SEQ ID NO: 54) (SEQ ID NO: 36)5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3′ 1-44 stop Eag Right (SEQ ID NO: 37)5′-CGGCCGTCAAGCGCGAGCTTCTCTCAGGCG-3′

This amplimer was cloned into pGemT and screened for inserts in the T7(5′) orientation.

Construction of (G₄S)₂-AD1(“(G₄S)₂” disclosed as SEQ ID NO: 55)

A duplex oligonucleotide, designated (G₄S)₂-AD1 (“(G₄S)₂” disclosed asSEQ ID NO: 55), was synthesized (Sigma Genosys) to code for the aminoacid sequence of AD1 preceded by 11 residues of the linker peptide withthe first two codons comprising a BamHI restriction site. A stop codonand an EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO: 38)

Two complimentary overlapping oligonucleotides, designated AKAP-IS Topand AKAP-IS Bottom, were synthesized.

AKAP-IS Top (SEQ ID NO: 22)5′GGATCCGGAGGTGGCGGGTCTGGCGGAGGTGGCAGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCTGACGGCCG- 3′ AKAP-IS Bottom (SEQID NO: 23) 5′CGGCCGTCAGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCTGCCACCTCCGCCAGACCCGCCACCTCCGGATCC- 3′

The duplex was amplified by PCR using the following primers:

G4S Bam-Left (“G4S” disclosed as SEQ ID NO: 54) (SEQ ID NO: 24)5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3′ AKAP-IS stop Eag Right (SEQ ID NO:25) 5′-CGGCCGTCAGGCCTGCTGGATG-3′

This amplimer was cloned into the pGemT vector and screened for insertsin the T7 (5′) orientation.

Ligating DDD1 with CH1

A 190 by fragment encoding the DDD1 sequence was excised from pGemT withBamHI and NotI restriction enzymes and then ligated into the same sitesin C_(H)1-pGemT to generate the shuttle vector C_(H)1-DDD1-pGemT.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from pGemTwith BamHI and NotI and then ligated into the same sites in CH1-pGemT togenerate the shuttle vector CH1-AD1-pGemT.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either C_(H)1-DDD1 or CH1-AD1 can beincorporated into any IgG-pdHL2 vector. The entire heavy chain constantdomain is replaced with one of the above constructs by removing theSacII/EagI restriction fragment (C_(H)1-C_(H)3) from pdHL2 and replacingit with the SacII/EagI fragment of C_(H)1-DDD1 or C_(H)1-AD1, which isexcised from the respective pGemT shuttle vector.

CH1-DDD2-Fab-hR1-pdHL2

C_(H)1-DDD2-Fab-hR1-pdHL2 is an expression vector for production ofC_(H)1-DDD2-Fab-hR1, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd via a 14amino acid residue Gly/Ser peptide linker.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide (GGGGSGGGCG, SEQ ID NO:26) and residues 1-13of DDD2, were made synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

G4S-DDD2 top (“G4S” disclosed as SEQ ID NO: 54) (SEQ ID NO: 27)5′GATCCGGAGGTGGCGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3′ G4S-DDD2 bottom (“G4S” disclosed as SEQ IDNO: 54) (SEQ ID NO: 28)5′GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTCCGC CAGACCCGCCACCTCCG-3′

The duplex DNA was ligated with the shuttle vector CH1-DDD1-pGemT, whichwas prepared by digestion with BamHI and PstI, to generate the shuttlevector CH1-DDD2-pGemT. A 507 by fragment was excised from CH1-DDD2-pGemTwith SacII and EagI and ligated with the IgG expression vector hR1pdHL2,which was prepared by digestion with SacII and EagI. The finalexpression construct is C_(H)1-DDD2-Fab-hR1-pdHL2.

Generation of C_(H)1-AD2-Fab-h679-pdHL2

C_(H)1-AD2-Fab-h679-pdHL2 is an expression vector for the production ofC_(H1)-AD2-Fab-h679 and is useful as a template for the DNA sequenceencoding AD2. The expression vector is engineered as follows. Twooverlapping, complimentary oligonucleotides (AD2 Top and AD2 Bottom),which comprise the coding sequence for AD2 and part of the linkersequence, are made synthetically. The oligonucleotides are annealed andphosphorylated with T4 polynucleotide kinase, resulting in overhangs onthe 5′ and 3′ ends that are compatible for ligation with DNA digestedwith the restriction endonucleases BamHI and SpeI, respectively.

AD2 Top (SEQ ID NO: 29)5′GATCCGGAGGTGGCGGGTCTGGCGGATGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCGGCTGCTGAA-3′ AD2 Bottom (SEQ ID NO:30) 5′TTCAGCAGCCGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCCACATCCGCCAGACCCGCCACCTCCG-3′

The duplex DNA is ligated into the shuttle vector C_(H)1-AD1-pGemT,which is prepared by digestion with BamHI and SpeI, to generate theshuttle vector CH1-AD2-pGemT. A 429 base pair fragment containing C_(H)1and AD2 coding sequences is excised from the shuttle vector with SacIIand EagI restriction enzymes and ligated into h679-pdHL2 vector that isprepared by digestion with those same enzymes, resulting inC_(H)1-AD2-Fab-h679-pdHL2.

Generation of C_(H)3-AD2-IgG-pdHL2 for expressing C_(H3)-AD2-IgG

C_(H)3-AD2-IgG modules have an AD2 peptide fused to the carboxylterminus of the heavy chain of IgG via a 9 amino acid residue peptidelinker. The DNA coding sequences for the linker peptide (GSGGGGSGG, SEQID NO:31) followed by the AD2 peptide (CGQIEYLAKQIVDNAIQQAGC, SEQ IDNO:18) are coupled to the 3′ end of the C_(H)3 (heavy chain constantdomain 3) coding sequence by standard recombinant DNA methodologies,resulting in a contiguous open reading frame. When the heavy chain-AD2polypeptide is co-expressed with a light chain polypeptide, an IgGmolecule is formed possessing two AD2 peptides, which can therefore bindtwo Fab-DDD2 dimers. The C_(H)3-AD2-IgG module can be combined with anyC_(H)1-DDD2-Fab module to generate a wide variety of hexavalentstructures composed of an Fc fragment and six Fab fragments. If theC_(H)3-AD2-IgG module and the C_(H)1-DDD2-Fab module are derived fromthe same parental monoclonal antibody (MAb) the resulting complex ismonospecific with 6 binding arms to the same antigen. If the modules areinstead derived from two different MAbs then the resulting complexes arebispecific, with two binding arms for the specificity of theC_(H)3-AD2-IgG module and 4 binding arms for the specificity of theC_(H)1-DDD2-Fab module.

A plasmid shuttle vector was produced to facilitate the conversion ofany IgG-pdHL2 vector into a C_(H)3-AD2-IgG-pdHL2 vector. The gene forthe Fc (C_(H)2 and C_(H)3 domains) was amplified using the pdHL2 vectoras a template and the oligonucleotides Fc BgIII Left and Fc Bam-EcoRIRight as primers.

Fc BglII Left (SEQ ID NO: 32) 5′-AGATCTGGCGCACCTGAACTCCTG-3′ FcBam-EcoRI Right (SEQ ID NO: 33) 5′-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3′

The amplimer was cloned in the pGemT PCR cloning vector. The Fc insertfragment was excised from pGemT with XbaI and BamHI restriction enzymesand ligated with AD2-pdHL2 vector that was prepared by digestion ofC_(H)1-AD2-Fab-h679-pdHL2 with XbaI and BamHI, to generate the shuttlevector Fc-AD2-pdHL2.

To convert any IgG-pdHL2 expression vector to a C_(H)3-AD2-IgG-pdHL2expression vector, an 861 by BsrGI/NdeI restriction fragment is excisedfrom the former and replaced with a 952 by BsrGI/NdeI restrictionfragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the C_(H)3domain and NdeI cuts downstream (3′) of the expression cassette.

Generation of Hex-hR1

The DNL method is used to create Hex-hR1, a monospecific anti-IGF-1Rwith one Fc and six Fabs, by combining C_(H3)-AD2-IgGhR1 withC_(H)1-DDD2-Fab-hR1. Hex-hR1 is made in four steps.

Step 1, Combination: C_(H)1-DDD2-Fab-hR1 is mixed withC_(H)3-AD2-IgG-hR1 in phosphate buffered saline, pH 7.4 (PBS) with 1 mMEDTA, at a molar ratio of 4.2 such that there are twoC_(H)1-DDD2-Fab-hR1 for each AD2 on C_(H)3-AD2-IgG-hR1, allowing someexcess of C_(H)1-DDD2-Fab-hR1 to ensure that the coupling reaction iscomplete.

Step 2, Mild Reduction: Reduced glutathione (GSH) is added to a finalconcentration of 1 mM and the solution is held at room temperature(16-25° C.) for 1 to 24 hours.

Step 3, Mild Oxidation: Following reduction, oxidized glutathione (GSSH)is added directly to the reaction mixture to a final concentration of 2mM and the solution is held at room temperature for 1 to 24 hours.

Step 4, Isolation of the DNL product: Following oxidation, the reactionmixture is loaded directly onto a Protein-A affinity chromatographycolumn. The column is washed with PBS and the Hex-hR1 eluted with 0.1 MGlycine, pH 2.5. The unreacted C_(H)1-DDD2-Fab-hR1 is removed from thedesired product in the unbound fraction. Other hexavalent DNL constructscan be prepared similarly by mixing a selected pair of C_(H)3-AD2-IgGand C_(H)1-DDD2-Fab.

A list of such DNL constructs and structural controls related to thepresent invention is provided in Table 4. Each of these constructs wasshown to retain the binding activities of the constitutive antibodies.

TABLE 4 hR1-containing DNL constructs and structural controls ValencyDNL code IgG-AD2 Fab-DDD2 2 4 6 Hex-hR1 hR1 hR1 — — IGF-1R Hex-hRS7 hRS7hRS7 — — EGP-1 Hex-hPAM4 hPAM4 hPAM4 — — MUC1 Hex-hMN-14 hMN-14 hMN14 —— CEACAM5 Hex-hLL1 hLL1 hLL1 — — CD74 Hex-hL243 hL243 hL243 — — HLA-DR1R-E1 hR1 hRS7 IGF-1R EGP-1 — 1R-14 hR1 hMN-14 IGF-1R CEACAM5 — 1R-15hR1 hMN-15 IGF-1R CEACAM6 — 1R-31 hR1 hAFP IGF-1R AFP — 1R-74 hR1 hLL1IGF-1R CD74 — 1R-C2 hR1 hL243 IGF-1R HLA-DR — 1R-M1 hR1 hPAM4 IGF-1RMUC1 E1-1R hRS7 hR1 EGP-1 IGF-1R — M1-1R hPAM4 hR1 MUC1 IGF-1R — 14-1RhMN-14 hR1 CEACAM5 IGF-1R — 74-1R hLL1 hR1 CD74 IGF-1R — C2-1R hL243 hR1HLA-DR IGF-1R — 22-20 hLL2 hA20 CD22 CD20 —

Example 7 IGF-1R Expression in Cancer Cell Lines

Zenon-labeled various parental antibodies as well as multivalentantibodies derived from these antibodies were used to assess theexpression levels of cognate antigens in several cancer cell lines byflow cytometry performed on Guava instrument. Expression of IGF-1R wasconfirmed by the binding of hR1 to MCF-7 (breast cancer), CaPan1(pancreatic cancer), and DU-145 (prostate cancer), as shown in FIG. 10.The dual expression of IGF-1R and AFP in HepG2 (liver cancer) was alsoshown in FIG. 11 by the binding of humanized anti-AFP IgG and TF 18(made by combining C_(H)1-DDD2-Fab-hAFP with C_(H)1-AD2-Fab-h679 tocontain two Fab fragments of hAFP), as well as by the enhanced bindingof hR1-IgG-AD2 (the dimer of C_(H)3-AD2-IgG-hR1) and 1R-31, suggesting ahigher affinity of these multivalent DNL constructs. The expression ofCEACAM6 in Hep G2 was noted by the observation of the enhanced bindingof 1R-15. Additional studies performed with MCF-7, DU-145, and ME-180(cervical cancer) on FACScan are presented in FIG. 12 and summarized inTable 5, which corroborate the findings by Guava that the multivalentDNL constructs exhibit enhanced binding to target cell lines compared totheir parental antibodies. Interestingly, the multivalent, bispecificconstructs appear to bind more avidly than their multivalent,monospecific counterparts in cell lines expressing differential levelsof relevant antigens.

TABLE 5 Flow cytometry data obtained from FACScan MCF-7 DU145 ME-180Antibody MFI % Positive Antibody MFI % Positive Antibody MFI % Positive/ / — 2.4 1.96 — 1.86 2 human IgG 1.84 2.34 human IgG 2.21 1.44 humanIgG 1.8 1.37 22-20 2.11 3.1 DNL1 2.74 7.88 DNL1 1.98 12.6 hR1 9.93 89.15hR1 5.33 30.39 hR1 3.65 10.74 hRS7 21.42 99.15 hRS7 10.58 82.82 hRS735.54 99.96 Hex-hR1 14.08 98.58 Hex-hR1 7.83 72.56 Hex-hR1 6.36 33.02Hex-hRS7 35.73 99.86 Hex-hRS7 17.03 93.74 Hex-hRS7 59.58 99.95 1R-E147.85 99.92 1R-E1 22.10 99.53 1R-E1 76.29 99.94 E1-1R 109.19 98.77 E1-1R53.96 99.9 E1-1R 254.8 99.89

Example 8 Neutralizing Activity of Hex-hR1 and 1R-E1

The following experiments were performed to determine the effect ofHex-hR1 or 1R-E1 on neutralizing the growth stimulating activity ofIGF-1 in DU-145 and ME-180, both of which express IGF-1R and EGP-1.Target cells were seeded at 2000/well onto 96-well plates and grownovernight in complete medium. Cells were washed twice with serum freemedium and exposed to a selected multivalent antibody at 0.8, 4, 20, and100 μg/mL in serum free medium for 2 h, followed by the addition ofIGF-1 to a final concentration of 10 ng/ml. Cells were incubated for 72hours and then subjected to MTS assay. Under these conditions, Hex-hR1suppressed the proliferation of DU-145 (FIG. 13A) and ME-180 (FIG. 13B)in a dose-dependent manner with statistical significance. Similarresults were obtained with 1R-E1 in ME-180 (FIG. 13C).

Example 9 Downregulation of IGF-1R

One major mechanism of anti-tumor actions induced by an anti-IGF-1Rantibody, despite its being an agonist or antagonist, is to downregulateIGF-1R via endocytosis leading to subsequent degradation in endosomalvesicles. As shown in FIG. 14, efficient downregulation of IGF-1R inMCF-7 or HT-29 (colorectal cancer) was clearly demonstrated with hR1 at100 nM as well as the two commercially available anti-IGF-1R antibodies(MAB391 and 24-60) serving as positive controls, but not with theanti-CD22 antibody, hLL2 (epratuzumab), which serves as a negativecontrol. Further studies revealed that Hex-hR1 and 1R-E1 were capable ofsubstantially reducing the level of IGF-1R at a concentration as low as0.1 nM in MCF-7, DU-145, and LnCap (androgen-dependent prostate cancer),as shown in FIGS. 15A and B.

Example 10 Blocking the Signaling Pathways Induced by IGF-1

Although hR1 may not appear to prevent the binding of IGF-1 tobead-immobilized rhIGF-1R, it effectively blocks IGF-1 from activatingvarious signaling molecules in three cell lines (MCF-7, DU-145, andME-180), as collectively shown in FIGS. 16 to 20, by the reduced levelsof phosphorylated IGF-1R (pIGF-1R), phosphorylated Akt (pAkt), andphosphorylated ERK1/2 (pERK1/2).

The described methods and compositions are of use for therapy ofprostate cancer. The Examples disclosed above provide in vitro results,showing hR1, as well as its hexavalent derivatives made by DNL (Hex-hR1,1R-E1, and E1-1R), can effectively downregulate IGF-1R and inhibit IGF-1from stimulating the proliferation of androgen-independent DU-145 cells.The higher potency observed for the DNL constructs is presumably due totheir enhanced avidity which may be further amplified for the bispecificcounterparts because the increase in targetable antigens on the cellsurface. As IGF-1R is expressed in various solid tumors and hematologicmalignancies, the skilled artisan will realize that the claimedcompositions and methods are also of use for therapy of other knownIGF-1R expressing cancers, such as multiple myeloma and hepatoma. Thecombination of hR1 with other antibodies, such as anti-EGFR(C225) oranti-HER2 (Herceptin), is also of therapeutic use, as discussed above.

Example 11 Effects of Various hR1 Constructs in the MCF-7 Breast CancerXenograft Model

Four-week old female athymic (nu/nu) mice are implanted with 60-dayrelease pellets of 0.5 mg 17β-estradiol (Sachdev et al., Cancer Res.2003; 63:627-35) and then injected with 10 million MCF-7 human breastcancer cells subcutaneously. When the tumors measure an average volumeof 200 mm³, groups of 9 mice are randomized for intraperitonealtreatment with the following agents, twice per week for 4 consecutiveweeks: (1) saline controls, same volume as test substances; (2) 400 μghR1IgG; (3) 800 μg hR1IgG; (4) 400 μg hRS7 IgG (anti-EGP-1); (5) 800 pghRS7 IgG; (6) 300 μg 1R-E1 hexavalent construct (hR1IgG-hRS7-4 Fab′ s);(7) 600 μg 1R-E1; and (8) 800 μg 1R-E1. Tumor volumes are measuredbidirectionally with a caliper twice weekly, beginning on the day ofrandomization and treatment; animal weights are also taken twice weekly.When mice become moribund or lose more than 20% of body weight, or whenthe tumors reach a size of 2.5 cm³, they are sacrificed, and tumors andnormal tissues removed and preserved in formalin for histological andimmunohistochemical analyses. By 60 days, the experiment is terminated,and shows continuous tumor growth of the controls, which are sacrificedas early as 5 weeks after therapy onset and then over the next 2 weeks.Evidence of inhibition of tumor growth is measured as 20% (relative tocontrols) for group 1, 35% for group 2, 54% for group 3, 11% for group4, 26% for group 5, 32% for group 6, 51% for group 7, and 68% for group8 at one week post therapy-end. These results indicate that the hR1antibody doses are more inhibitory of tumor growth than those for thehRS7 (anti-EGP-1) antibody, but which also shows some antitumoractivity, However, lower doses of the hexavalent bispecific antibodyconstruct of hR1 and hRS7, at relatively lower doses, show equivalent tohigher antitumor effects than the corresponding parental antibodies,suggesting greater potency for the bispecific antibody constructs madeby DNL. No treatment-related toxicities, particularly more than a 20%body weight loss, is observed in the treatment groups.

When the same experiment is repeated with similar MCF-7-bearing mice,using an irrelevant isotype control antibody instead of saline, andincluding hexavalent hR1 and hexavalent hRS7 groups at doses of 400 μgand 800 μg i.p. each, twice weekly for 4 weeks, tumor growth inhibitionis determined to range from 25-46% for hexavalent hR1 and 20-33% forhexavalent hRS7, suggesting that the hexavalent constructs are morepotent than their bivalent parental counterparts.

Example 12 Effects of hR1 Constructs in BxPC3 Human Pancreatic andColo205 Human Colonic Cancer Xenografts

Tumor xenografts in 5-6-week-old female nu/nu athymic mice are injecteds.c. with 2 million BxPC3 human pancreatic cancer or Colo205 humancolonic cancer cells mixed in Matrigel, and allowed to grow to about 200mm³ in size, and are randomized into groups of 11 each. Mice are treatedby i.p. injection twice weekly of saline vehicle (control) or testsubstances at various doses for 4 consecutive weeks. Tumors are measuredand the animals weighed and observed as per the prior Example. For eachof the tumor models, the following doses are given, with the percentagesof tumor growth-inhibition, G1 (comparing mean volumes of treated vs.control groups before more than 20% of the control mice are sacrificedbecause of advanced tumor growth) in parentheses:

BxPC3 Human Pancreatic Cancer Model

(1) 0.5 mg hR1 (39% GI)

(2) 1.0 mg hR1 (68% GI)

(3) 0.5 mg hPAM4 (15% GI)

(4) 1.0 mg hPAM4 (24% GI)

(5) 0.5 mg hRS7 (18% GI)

(6) 1.0 mg hRS7 (29% GI)

(7) 0.5 mg 1R-E1 (63% GI)

(8) 0.5 mg 1R-1M (48% GI)

(9) 1.0 mg hLL2 (anti-CD22 IgG) isotype control.

The results suggest a dose-dependent effect of hR1 in inhibiting growthof human pancreatic cancer xenografts, which appears superior to thehRS7 or hPAM4 antibodies, but the bispecific construct of hR1 and hRS7(1R-E1) appears to show enhanced activity over the same doses of theparental antibodies given separately, and somewhat less enhancedefficacy for the bispecific antibody of hR1 and hPAM4 (1R-1M).

Colo205 Human Colonic Cancer Model

(1) 0.5 mg hR1 (46% GI)

(2) 1.0 mg hR1 (70% GI)

(3) 0.5 mg hMN-14 (14% GI)

(4) 1.0 mg hMN-14 (29% GI)

(5) 0.5 mg 14-1R (58% GI)

(6) 1.0 mg 14-1R (83% GI)

(7) 1.0 mg hLL2 control.

A dose-dependent growth-inhibition is observed for both anti-IGFR1 andanti-CEACAM5 (hMN-14) humanized antibodies, with the former being morepotent in this model, but with the respective bispecific antibodyconstructs made by DNL showing improved efficacy compared to theequivalent doses of the bivalent parental antibodies.

Example 13 Effects of Effects of hR1 Constructs Alone and in Combinationwith Bortezomib in a Multiple Myeloma Xenografts Model

CAG human myeloma cells are grown in cell culture to a density thatallows 1 million cells to be harvested for transplantation to 6-8-weekold female SCID mice obtained from Charles River Laboratories(Frederick, Md.). The mice are immunosuppressed by pretreatment withfludarabine and cyclophosphamide 3 days before intravenous injection of5−10×16⁶ myeloma cells as described in Stein et al. (Blood. 2004;104:3705-11). Mice are examined daily for signs of distress or hind-legparalysis, and weighed weekly. Paralysis of the hind legs or a weightloss of >20% is used as the survival endpoint, when the animals areeuthanized. Groups of 8-10 mice are used. A dose-response study with hR1given i.p. twice weekly for 4 weeks at 100, 300, 600, and 1,000 μg hR1shows a significant (P<0.03) survival benefit compared to mice treatedwith the saline vehicle or with an unreactive isotype control antibody,which has a median survival of 40 days. The median survival of thehR1-treated mice ranged from 80 to 100 days, and shows a dose-response.The effects of combining bortezomib with hR1 are evaluated in the samemyeloma model. Treatments are given as two i.p. doses/week for 3 weeks,initiated on day 5 after injection of the myeloma cells. Given as asingle agent, 0.5 mg/kg bortezomib is well tolerated, with nobody-weight loss. Median survival in untreated control mice is 33 days,for bortezomib alone 40 days (21.2% increase). Treatment with hR1 at 0.6mg/mouse repeated twice weekly for 3 weeks increases the median survivaltime to 60 days. When bortezomib and hR1 treatments are combined, themedian survival time is increased further to 79 days for 0.5 mgbortezomib+0.6 mg hR1, which is significant (P=0.04). Therefore, anagent that is active in treating myeloma shows enhanced activity whencombined with this anti-IGFR1 antibody.

Example 14 Effects of Combination Therapy of ⁹⁰Y-hPAM4Radioimmunotherapy with Gemcitabine and anti-IGFR1 Antibody (hR1)Immunotherapy in Pancreatic Cancer

YS is a 61-year-old male diagnosed 2 months earlier with stage III/IV,metastatic, inoperable pancreatic adenocarcinoma, having a 6 cm diameterpancreatic lesion at the head of the pancreas and two metastases to theliver of about 3 and 4 cm in diameter. The patient has an elevatedCA19.9 titer of 7,200, but with most other laboratory values eitherborderline or within the normal range. He is active, but is easilyfatigued, has occasional abdominal pains requiring minimal medication,and has lost about 10 kg since diagnosis. He opts for an investigationaltreatment involving a 4-week therapy consisting of gemcitabine (GEMZAR®)given i.v. once weekly at 200 mg/m², ¹¹¹In-DOTA-hPAM4 antibody (labeledas described in Sharkey et al. [J Nucl Med. 2003 December;44(12):2000-18] given by infusion also on week 1 to assess antibodylocalization by immunoscintigraphy, followed by the next 3 consecutiveweekly infusions of 12 mCi/m² of ⁹⁰Y-DOTA-hPAM4 (labeled as per Sharkeyet al., ibid). On days 1, 7, 14, and 21, doses of hR1 of 10 mg/kg, 16mg/kg, 16 mg/kg, and 20 mg/kg are given by i.v. infusion. The patientexperiences some grade 1-2 nausea, back pain, hypotension, anorexia andfatigue following each infusion, reducing severity with each one, whichis mild because of being premedicated with 50 mg hydrocortisone,acetaminophen, and diphenylhydramine to control infusion reactions. At 4weeks post therapy, the patient undergoes FDG-PET and CT scanning tocompare the metabolism and size of the pancreatic cancer lesions beforeand after therapy, and blood is taken to measure the CA 19-9 tumorbiomarker titer. The SUV of the primary cancer changes to 3.3 from 8.9,and the two metastatic lesions in the liver shows a larger drop from 6.1and 5.3 to 5.3 and 3.5, respectively. CT measurements indicate a 1-cmreduction of the primary tumor, and an approximately 33% reduction inthe two liver metastases. At this time, the CA19.9 titer measures 930,representing a major drop from 7,200. Follow-up studies 4 weeks laterconfirm continued reductions of the SUV values and either stabilizationor a slight reduction of the sizes of the tumors, as measured by CT.Three months later, since the patient has stable disease, the therapy isrepeated, is tolerated well, and again shows stable disease, with noincrease of the CA19.9 titer at the next, 4-week, follow-up. It isconcluded that this combination therapy has, at the minimum, stabilizedthe disease, decreased the tumors' metabolic activity, and markedlyreduced the pancreatic cancer blood biomarker, CA19-9. The patient hasbeen normally active during this period, has no fatigue or pain, and hasgained back 4 kg body weight. There are no hematological or otherlaboratory abnormalities.

Example 15 Combination FOLFIRI Therapy of Metastatic Colorectal Cancerwith hR1

RS is a 71-year-old woman with no prior serious illness and presentingwith metastatic colonic cancer post resection of a sigmoid colon B3adenocarcinoma 6 months earlier. She refuses post-operative radiation orchemotherapy. Her blood CEA titer is elevated at 11 ng/ml. FDG-PET/CTimaging shows no recurrence at the primary resection, but 3 discretelesions (2-4 cm in diameter) in the right liver lobe and 1 larger lesion(6 cm) in the left liver lobe. Not being a candidate for salvage liverresection, she undergoes a combination of FOLFIRI combined with hR1therapy. On day 1, 180 mg/m² irinotecan is given in 500 ml normal salineas a 2-h infusion, and on days 1 and 2, 400 mg/m² of folinic acid isgiven as an i.v. bolus over 2 hours, followed by fluorouracil (2,400mg/m²) as a continuous 46-h infusion, every 2 weeks. Anti-IGF-1Rantibody, hR1, is given at 10 mg/kg as a slow infusion weekly ×2 weeks,including premedication as in the prior patient Example. Eight cycles ofthis combination therapy are given. Six weeks after completion oftherapy, FDG-PET/CT scans indicate a 60% reduction of size and also anSUV reduction of the left lobe metastasis, while 2 of 3 right lobemetastases appear about 1 cm in diameter while the third is unchanged.SUV values for 2 of the 3 are reduced to almost 0, and the third is 3.2.No change in circulating CEA is noted. After another 6 weeks, 2 of 3right lobe metastases are not visible, and the third is about 1.5 cm indiameter. The left lobe tumor now measures 3 cm in diameter. The patientis considered to be in a partial response, which is ongoing at 8 monthsfrom end of therapy.

Example 16 Therapy of a Patient with Hepatocelluar Carcinoma withRadiolabeled hR1 Monoclonal Antibody

A 57-year-old man presenting with jaundice, malaise, loss of weight, andgeneral weakness, is diagnosed with an inoperable hepatocellularcarcinoma that appears by computed tomography to extend about 6 cm indiameter in the right lobe of the liver, and to also appear as a single3-cm lesion in the left lobe. The right lobe lesion is confirmed bybiopsy to be hepatocellular carcinoma.

The patient is given two cycles of hR1 monoclonal antibody conjugated byDOTA with ⁹⁰Y, as described in Sharkey et al. (J Nucl Med. 2003December; 44(12):2000-18), so that an infusion is administered for eachtherapy dose of 25 mCi (100 mg antibody protein). The first therapy isgiven in an outpatient setting, and is repeated 6 weeks later. Prior toeach therapy, a diagnostic dose of ¹¹¹In conjugated by DOTA to theantibody (labeling also described in Sharkey et al, 2003, ibid), is alsoinjected in order to demonstrate tumor targeting and to estimate theradiation dose delivered to the tumor and to other normal tissues, suchas liver, kidney and bone marrow, so that the therapeutic dose with ⁹⁰Y,given a week later, can be adjusted so as not to induce normaltissue/organ toxicity beyond what is considered tolerable (e.g., 2000cGy to kidneys). The patient is then monitored for response by repeatedcomputer tomography (CT) scans every 4-8 weeks post therapy, as well asby serum AFP, bilirubin, transaminase, and LDH levels.

Eight weeks after the second therapeutic administration of the⁹⁰Y-labeled antibody, his serum levels of bilirubin, transaminases, andLDH decrease to about 20% above normal, and his serum AFP titer ismeasured at 60 ng/mL, which also constitutes an improvement. CTmeasurements of his liver disease show an almost complete disappearanceof the left lobe lesion and a 40% reduction of the larger mass in theright lobe. The patient then becomes a candidate for surgical resectionof his right lobe, since it is considered that the remaining smalllesion in the left lobe is not cancer, but scar tissue.

Example 17 Therapy of a Patient with ⁹⁰Y-labeled hR1Antibody Combinedwith Naked hR1Antibody

A 62-year-old man has a history of Dukes' C rectal carcinoma that isresected 3 years earlier, followed by radiation therapy and5-fluorouracil/folinic acid chemotherapy. The patient begins to show arise in his plasma CEA titer, reaching a level of 30 ng/mL. The patientundergoes various diagnostic procedures because of a suspectedrecurrence. It is found, by computed tomography, that there are twometastases present in his liver, one being 3 cm in diameter in his rightlobe, and the other being somewhat smaller in the left lobe, close tothe interlobe ligament. The patient is first given 3 weekly infusions of10 mg/kg hR1 antibody, followed by a dose of 25 mCi ⁹⁰Y conjugated tohR1 antibody, given at a protein dose of 50 mg by intravenous infusionover a period of 2 hours, on the third week of naked hR1 therapy, priorto the third hR1 injection. This therapy is repeated two months later.The patient shows a drop of his white blood cells and platelets,measured 2-4 weeks after the last therapy infusion, but recuperates atthe 8-week post-therapy evaluation. The computed tomography findings at3 months post-therapy reveal 40% shrinkage of the major tumor metastasisof the right liver lobe, and a lesser reduction in the left-lobe tumor.

At the 6-month follow-up, his tumor lesions have been reduced, intwo-diameter CT-measurements, by about 70 percent, his plasma CEA is at8 ng/mL, and his general condition is improved, with no apparenttoxicity or adverse events related to the therapy.

Example 18 Treatment of a Breast Cancer Patient with Y-90 hR1MAb andwith Naked hR1MAb

A 56-year-old woman with a history of recurrent adencarcinoma of thebreast presents with cervical lymph node and left lung metastases. Sherelapses twice after chemotherapy and hormonal therapies. She is thengiven three therapeutic injections, each one week apart, of⁹⁰Y-conjugated hR1MAb i.v., at a dose of 15 mCi ⁹⁰Y each in a proteindose of antibody of 100 mg. Four weeks after therapy, her white bloodcell and platelet counts decrease by approximately 50%, but recuperateby 9 weeks post-therapy. At a restaging 12 weeks post-therapy, anapproximately 30% decrease in pulmonary and nodal metastases is measuredby computed tomography. Thereafter, she receives 4 weekly infusions,over 4 hours each, of naked hR1 antibody, which is tolerated well,except for some transient rigors and chills, and without any adverseeffects on her blood counts or blood chemistries. The naked antibodydose for each infusion is 12 mg/kg. Approximately 8 weeks later,restaging by computed tomography indicates an additional decrease inmeasurable lesions by about 20 percent. At the followup examination 3months later, her disease appears to be stable (i.e., no evidence ofadditional or progressive growth).

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and used without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the METHODS described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. An anti-IGF-1R (insulin-like growth factor type 1 receptor) antibodyor antigen-binding fragment thereof that binds to an epitope of IGF-1Rcomprising the first half of the cysteine-rich domain of IGF-1R, betweenamino acid residues 151 and 222 of human IGF-1R.
 2. The anti-IGF-1Rantibody or fragment thereof of claim 1, wherein said antibody exhibitsat least one functional characteristic selected from the groupconsisting of: (i) binds to human IGF-1R and does not bind to humaninsulin receptor (IR); (ii) is not an agonist of IGF-1R; (iii) does notblock binding of IGF-1 or IGF-2 to an isolated IGF-1R; (iv) neutralizesthe activation of IGF-1R by IGF-1 in intact cells; and (v) blocksbinding to IGF-1R of an hR1 antibody comprising the variable regionsequences SEQ ID NO:9 and SEQ ID NO:10.
 3. The anti-IGF-1R antibody orfragment thereof of claim 1, wherein said anti-IGF-1R antibody does notcompete for binding to isolated IGF-1R with the anti-IGF-1R antibodies24-31, 24-57, 17-69, 1-2, 1H7, 2C8 or 3B7.
 4. The anti-IGF-1R antibodyor fragment thereof of claim 1, wherein said anti-IGF-1R antibody doesnot bind to amino acid residues 1-150, 223-274, 184-283, 283-440,440-514, 514-586 or 1323-1337 of isolated human IGF-1R.
 5. Theanti-IGF-1R antibody or fragment thereof of claim 1, wherein saidanti-IGF-1R antibody comprises the heavy chain variable regioncomplementarity determining region (CDR) sequences CDR1 (DYYMY, SEQ IDNO:1), CDR2 (YITNYGGSTYYPDTVKG, SEQ ID NO:2) and CDR3 (QSNYDYDGWFAY, SEQID NO:3) and the light chain variable region CDR sequences CDR1(KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQ ID NO:5) and CDR3(QQYSNYPLT, SEQ ID NO:6).
 6. An anti-IGF-1R antibody or antigen-bindingfragment thereof that blocks binding to isolated human IGF-1R of an R1antibody comprising the heavy chain variable region CDR sequences CDR1(DYYMY, SEQ ID NO:1), CDR2 (YITNYGGSTYYPDTVKG, SEQ ID NO:2) and CDR3(QSNYDYDGWFAY, SEQ ID NO:3) and the light chain variable region CDRsequences CDR1 (KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQ ID NO:5)and CDR3 (QQYSNYPLT, SEQ ID NO:6).
 7. The anti-IGF-1R antibody orfragment thereof of claim 6, wherein said anti-IGF-1R antibody binds toan epitope of IGF-1R comprising the first half of the cysteine-richdomain of IGF-1R, between amino acid residues 151 and 222 of humanIGF-1R.
 8. The anti-IGF-1R antibody or fragment thereof of claim 6,wherein said antibody exhibits at least one functional characteristicselected from the group consisting of: (i) binds to human IGF-1R anddoes not bind to human insulin receptor (IR); (ii) is not an agonist ofIGF-1R; (iii) does not block binding of IGF-1 or IGF-2 to an isolatedIGF-1R; and (iv) neutralizes the activation of IGF-1R by IGF-1 in intactcells.
 9. The anti-IGF-1R antibody or fragment thereof of claim 6,wherein said antibody or fragment is a naked antibody or fragment or isconjugated to at least one diagnostic or therapeutic agent.
 10. Ananti-IGF-1R antibody or antigen-binding fragment thereof comprising theheavy chain variable region CDR sequences CDR1 (DYYMY, SEQ ID NO:1),CDR2 (YITNYGGSTYYPDTVKG, SEQ ID NO:2) and CDR3 (QSNYDYDGWFAY, SEQ IDNO:3) and the light chain variable region CDR sequences CDR1(KASQEVGTAVA, SEQ ID NO:4), CDR2 (WASTRHT, SEQ ID NO:5) and CDR3(QQYSNYPLT, SEQ ID NO:6).
 11. The anti-IGF-1R antibody or fragmentthereof of claim 10, wherein said antibody exhibits at least onefunctional characteristic selected from the group consisting of: (i)binds to human IGF-1R and does not bind to human insulin receptor (IR);(ii) is not an agonist of IGF-1R; (iii) does not block binding of IGF-1or IGF-2 to an isolated IGF-1R; (iv) neutralizes the activation ofIGF-1R by IGF-1 in intact cells; (v) blocks binding to IGF-1R of an hR1antibody comprising the variable region sequences SEQ ID NO:9 and SEQ IDNO:10; and (vi) binds to an epitope of IGF-1R comprising the first halfof the cysteine-rich domain of IGF-1R, between amino acid residues 151and 222 of human IGF-1R.
 12. The anti-IGF-1R antibody or fragmentthereof of claim 10, wherein said anti-IGF-1R antibody is a murineantibody, a chimeric antibody, a humanized antibody or a human antibody.13. The anti-IGF-1R antibody or fragment thereof of claim 12, whereinsaid anti-IGF-1R antibody is a humanized antibody comprising frameworkand constant region sequences from a human antibody.
 14. The anti-IGF-1Rantibody or fragment thereof of claim 13, wherein said humanizedanti-IGF-1R antibody is a humanized R1 (hR1) antibody comprising theamino acid sequences of SEQ ID NO:9 (hR1VH) and SEQ ID NO:10 (hR1VK).15. The anti-IGF-1R antibody or fragment thereof of claim 13, whereinthe variable region sequences of said humanized anti-IGF-1R antibodyhave at least 90%, at least 95%, at least 98% or at least 99% sequencehomology to SEQ ID NO:9 and SEQ ID NO:10.
 16. The anti-IGF-1R antibodyor fragment thereof of claim 13, wherein the variable region sequencesof said humanized anti-IGF-1R antibody comprise the amino acid sequencesof SEQ ID NO:9 and SEQ ID NO:10 except for 20 or fewer conservativeamino acid substitutions in the sequences of SEQ ID NO:9 and SEQ IDNO:10.
 17. The anti-IGF-1R antibody or fragment thereof of claim 13,wherein said anti-IGF-1R antibody is a chimeric R1 (cR1) antibodycomprising the amino acid sequences of SEQ ID NO:7 (R1VH) and SEQ IDNO:8 (R1VK) attached to human antibody constant region sequences. 18.The anti-IGF-1R antibody or fragment thereof of claim 13, wherein thevariable region sequences of said chimeric anti-IGF-1R antibody have atleast 90%, at least 95%, at least 98% or at least 99% sequence homologyto SEQ ID NO:7 and SEQ ID NO:8.
 19. The anti-IGF-1R antibody or fragmentthereof of claim 13, wherein the variable region sequences of saidchimeric anti-IGF-1R antibody comprise the amino acid sequences of SEQID NO:7 and SEQ ID NO:8 except for 20 or fewer conservative amino acidsubstitutions in the sequences of SEQ ID NO:7 and SEQ ID NO:8.
 20. Theanti-IGF-1R antibody or fragment thereof of claim 10, wherein saidantibody is a naked antibody.
 21. The anti-IGF-1R antibody or fragmentthereof of claim 10, wherein said antibody is attached to (i) at leastone therapeutic agent, (ii) at least one diagnostic agent, or (iii) atleast one therapeutic agent and at least one diagnostic agent.
 22. Theanti-IGF-1R antibody or fragment thereof of claim 21, wherein saidtherapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, an anti-angiogenic agent, a cytokine,a chemokine, a growth factor, a hormone, a drug, a prodrug, an enzyme,an oligonucleotide, a pro-apoptotic agent, an interference RNA, aphotoactive therapeutic agent, a cytotoxic agent, a chemotherapeuticagent and a toxin.
 23. The anti-IGF-1R antibody or fragment thereof ofclaim 21, wherein said diagnostic agent is selected from the groupconsisting of a radioisotope, a dye, a radiological contrast agent, anultrasound contrast agent, a fluorescent label, a chemiluminescentlabel, an enzyme, an enhancing agent and a paramagnetic ion.
 24. Theanti-IGF-1R antibody of claim 10, wherein said antibody comprisesconstant region sequences of a human IgG1 or IgG4 antibody.
 25. A fusionprotein comprising the anti-IGF-1R antibody or fragment thereof ofclaim
 1. 26. A multispecific antibody comprising the anti-IGF-1Rantibody or fragment thereof of claim 1 attached to at least one otherantibody or fragment thereof.
 27. The multispecific antibody of claim26, wherein the other antibody binds to a tumor-associated antigen. 28.The multispecific antibody of claim 27, wherein the tumor-associatedantigen is selected from the group consisting of carbonic anhydrase IX,CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, CD20, IGF-1R, CD21, CD22, CD23, CD25, CD29,CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95,CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, B7, ED-Bfibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1,hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1(ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5,PAM4 antigen, NCA-95, NCA-90, PSMA, EGP-1, EGP-2, AFP, Ia, HM1.24,HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAILreceptor (R1 and R2), VEGFR, EGFR, PIGF, complement factors C3, C3a,C3b, C5a, C5, and an oncogene product.
 29. The multispecific antibody ofclaim 26, wherein the other antibody binds to a hapten on a targetableconstruct.
 30. The multispecific antibody of claim 26, wherein the otherantibody is a chimeric, humanized or human antibody.
 31. Themultispecific antibody of claim 26, wherein the other antibody isselected from the group consisting of the hPAM4, hA20, hA19, hIMMU31,hLL1, hLL2, hMu-9, hL243, hMN-14, hMN-15, hMN-3, hRS7, h679 and h734antibodies.
 32. The multispecific antibody of claim 26, wherein themultispecific antibody is a bispecific antibody.
 33. A method ofdiagnosing or treating cancer comprising administering to an individualwith a cancer that expresses IGF-1R an anti-IGF-1R antibody or antigenbinding fragment thereof according to claim
 1. 34. The method of claim33, wherein the anti-IGF-1R antibody is a chimeric, humanized or humanantibody.
 35. The method of claim 33, wherein the anti-IGF-1R antibodycomprises the heavy chain CDR sequences CDR1 (DYYMY, SEQ ID NO:1), CDR2(YITNYGGSTYYPDTVKG, SEQ ID NO:2) and CDR3 (QSNYDYDGWFAY, SEQ ID NO:3)and the light chain CDR sequences CDR1 (KASQEVGTAVA, SEQ ID NO:4), CDR2(WASTRHT, SEQ ID NO:5) and CDR3 (QQYSNYPLT, SEQ ID NO:6).
 36. The methodof claim 33, wherein the anti-IGF-1R antibody is a humanized R1 antibodycomprising the amino acid sequences of SEQ ID NO:9 (hR1VH) and SEQ IDNO:10 (hR1 VK).
 37. The method of claim 33, wherein said anti-IGF-1Rantibody is a naked antibody.
 38. The method of claim 37, wherein themethod is a method of treating cancer and the method further comprisesadministering to the individual at least one other therapeutic agent.39. The method of claim 38, wherein the at least one other therapeuticagent is selected from the group consisting of a radionuclide, animmunomodulator, an anti-angiogenic agent, a cytokine, a chemokine, agrowth factor, a hormone, a drug, a prodrug, an enzyme, anoligonucleotide, an interference RNA, a pro-apoptotic agent, aphotoactive therapeutic agent, a cytotoxic agent, a chemotherapeuticagent, an antibody, an antigen-binding antibody fragment and a toxin.40. The method of claim 39, wherein the at least one other therapeuticagent is selected from the group consisting of an EGFR inhibitor,erlotinib, an anti-EGFR antibody, an IGF-1R inhibitor, a tryphostin,AG1024, AG538, a pyrrolo[2,3-d]-pyrimidine derivative, NVP-AEW541 and asecond anti-IGF-1R antibody.
 41. The method of claim 39, wherein the atleast one other therapeutic agent is selected from the group consistingof 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines,bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors,irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans,cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, estramustine, epidophyllotoxin, estrogen receptor bindingagents, etoposide (VP16), etoposide glucuronide, etoposide phosphate,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, gemcitabine,hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide,leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, navelbine, nitrosurea, plicomycin, procarbazine, paclitaxel,pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen,taxol, temazolomide, DTIC, transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.
 42. The method of claim33, wherein the method is a method of treating cancer and saidanti-IGF-1R antibody is attached to at least one therapeutic agent. 43.The method of claim 33, wherein the method is a method of diagnosingcancer and said anti-IGF-1R antibody is attached to at least onediagnostic agent.
 44. The method of claim 43, wherein said diagnosticagent is selected from the group consisting of a radioisotope, a dye, aradiological contrast agent, an ultrasound contrast agent, a fluorescentlabel, a chemiluminescent label, an enzyme, an enhancing agent and aparamagnetic ion.
 45. The method of claim 43, wherein said anti-IGF-1Rantibody or fragment thereof is part of a fusion protein or a bispecificantibody.
 46. The method of claim 39, wherein the immunomodulator isselected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, IL-25, interferon-alpha, interferon-beta,interferon-gamma, TNF-alpha and the stem cell growth factor designated“S1 factor.
 47. The method of claim 33, wherein the cancer is selectedfrom the group consisting of Wilms' tumor, Ewing sarcoma, neuroblastoma,neuroendocrine tumors, melanoma, glioblastoma, breast, colon, rectal,gastric, prostate, liver, renal, biliary, pancreatic, lung, endometrial,cervical, ovarian, esophageal, medullary thyroid, bladder,head-and-neck, skin cancer, acute lymphoblastic leukemia, acutemyelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenousleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma,astrocytoma and glioma.
 48. A method of treating cancer comprisingadministering to an individual with a cancer that expresses IGF-1R ananti-IGF-1R antibody or antigen binding fragment thereof according toclaim
 6. 49. A method of treating premalignant or dysplastic lesionscomprising administering to an individual with a premalignant ordysplastic lesion that expresses IGF-1R an anti-IGF-1R antibody orantigen binding fragment thereof according to claim
 10. 50. A method oftreating cancer comprising administering to an individual with a cancerthat expresses IGF-1R an anti-IGF-1R antibody or antigen bindingfragment thereof according to claim
 10. 51. An isolated nucleic acidencoding an anti-IGF-1R antibody according to claim
 1. 52. The isolatednucleic acid of claim 51, encoding the sequences of SEQ ID NO:9 and SEQID NO:10.
 53. An expression vector comprising an isolated nucleic acidaccording to claim
 51. 54. A host cell comprising an expression vectoraccording to claim
 53. 55. A method of producing an anti-IGF-1R antibodyor fragment thereof comprising: a) obtaining a host comprising anexpression vector according to claim 53; and b) incubating the host cellin medium to express an anti-IGF-1R antibody or fragment.